Methods for detection of gram negative bacteria

ABSTRACT

Method for the determination of chlamydial or gram negative bacterial antigen comprising contacting a sample potentially containing extracted antigen with an optically active surface comprising an attachment layer, and a layer of non-specific protein.

RELATED APPLICATIONS

This is a division of application Ser. No. 08/075,952, filed Jun. 10,1993, now U.S. Pat. No. 5,541,057 hereby incorporated by reference inits totality (including drawings), which is a continuation-in-part ofU.S. patent applications: Garret Moddel et al., U.S. application Ser.No. 07/924,343, filed Jul. 31, 1992, now abandoned Garret Moddel et al.,U.S. application Ser. No. 07/873,097, filed Apr. 24, 1992, abandonedwhich is a continuation-in-part application of Garrett Moddel et al.,U.S. application Ser. No. 07/408,291, filed Sep. 18, 1989, nowabandoned. All of the above noted applications (including drawings) aremade a part hereof, and are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to devices which produce a detectableattenuation of the spectral characteristic of light impinging on thedevice by thin film phenomenon.

BACKGROUND OF THE INVENTION

Sandstrom et al., 24 Applied Optics 472, 1985, describe use of anoptical substrate of silicon with a layer of silicon monoxide and alayer of silicon dioxide formed as dielectric films. They indicate thata change in film thickness changes the properties of the opticalsubstrate to produce different colors related to the thickness of thefilm. That is, the thickness of the film is related to the colorobserved and a film provided on top of an optical substrate may producea visible color change. They indicate that a mathematical model can beused to quantitate the color change, and that " c!alculations performedusing the computer model show that very little can be gained in opticalperformance from using a multilayer structure . . . but a biolayer onthe surface changes the reflection of such structures very little sincethe optical properties are determined mainly by the interfaces insidethe multilayer structure . . . The conclusion is, somewhat surprisingly,that the most sensitive system for detection of biolayers is a singlelayer coating, while in most other applications performance can beimproved by additional dielectric layers."

Sandstrom et al., go on to indicate that slides formed from metal oxideson metal have certain drawbacks, and that the presence of metal ions canalso be harmful in many biochemical applications. They indicate that theideal top dielectric film is a 2-3 nm thickness of silicon dioxide whichis formed spontaneously when silicon monoxide layer is deposited inambient atmosphere, and that a 70-95 nm layer of silicon dioxide on a40-60 nm layer of silicon monoxide can be used on a glass or plasticsubstrate. They also describe formation of a wedge of silicon monoxideby selective etching of the silicon monoxide, treatment of the silicondioxide surface with dichlorodimethylsilane, and application of abiolayer of antigen and antibody. From this wedge construction they wereable to determine film thickness with an ellipsometer, and note that the"maximum contrast was found in the region about 65 nm where theinterference color changed from purple to blue." They indicate that thesensitivity of such a system is high enough for the detection of proteinantigen by immobilized antibodies. They conclude "the designs given aresensitive enough for a wide range of applications. The materials, i.e.,glass, silicon, and silicon oxides, are chemically inert and do notaffect the biochemical reaction studied. Using the computations above itis possible to design slides that are optimized for differentapplications. The slides can be manufactured and their quality ensuredby industrial methods, and two designs are now commercially available.It is our hope that these sensitive, versatile, and inexpensive toolswill further the development of simplified methods in immunology andbiochemistry." Citation omitted.!

Nygren et al., 59 J. Immunol. Methods 145, 1983, describe a systemsimilar to that described above, in which specific anti-human serumalbumin (HSA) antibodies are used to detect HSA. FIG. 2 of thispublication indicates that 10⁻⁵ mg/ml of HSA can be detected with a 16hour incubation, but that 10⁻⁶ mg/ml could not be detected in thissystem. They also state " a!fter 72 hour of incubation time, thedetection limit was lower (down to 1 ng/ml), however, the reaction wasthen more sensitive to unspecific reactions, giving rise to eventuallyoccurring positive controls."

Nygren et al., U.S. Pat. No. 4,558,012 describe a similar system exceptthat the overall array of layers is adapted to reduce reflection inrespect to non-monochromatic or white light in the wavelength range of525-600 nM.

SUMMARY OF THE INVENTION

This invention features improved devices, and methods for using suchdevices, for detecting the presence or amount of an analyte of interestwithin a sample. In contrast to prior devices, those of the presentinvention allow detection of extremely small quantities of analyte in asample, in amounts as low as 0.1 nM, 0.1 ng/ml, or 2×10³ organisms oreven as low as 50 fg in a rapid assay lasting only a few minutes. Totalassay times may vary from one hour to a few minutes from the initiationof the assay protocol (i.e., from the time that the analyte containingsample is contacted with the device). Indeed, the devices of the presentinvention permit detection of over 30% more true positive samples thanprior devices and methods have previously permitted in certain assays,such as an assay for Streptococcus A antigen. The invention is basedupon the finding of better structures in the present device compared tothose described by Sandstrom and co-workers (see above), the details ofwhich are provided below. Such devices can be used in an instrumentedformat. They are also useful when provided in a format in which a visualcolor change can be observed, especially when that visual color changeis extremely easy to interpret, e.g., as a change from a gold backgroundto a dark purple or blue color.

Thus, in a first aspect, the invention features a device for detectingthe amount or presence of an analyte of interest. The device includes asubstrate which has an optically active surface exhibiting a first colorin response to light impinging thereon. This first color is defined as aspectral distribution of the emanating light. The substrate alsoexhibits a second color which is different from the first color (byhaving a combination of wavelengths of light which differ from thatcombination present in the first color, or having a different spectraldistribution, or by having an intensity of one or more of thosewavelengths different from those present in the first color). The secondcolor is exhibited in response to the same light when the analyte ispresent on the surface. Such a device provides a sensitive method fordetecting an amount of 0.1 ng, 0.1 nM, 0.1 ng/ml, 50 fg, or 2×10³organisms containing the analyte of interest. Indeed, in preferredembodiments, the amount detected can be considerably smaller by as muchas 10, 100 or even 1000 fold. The change from one color to another canbe measured either by use of an instrument, or by eye. Such sensitivedetection is a significant advance over the devices described bySandstrom and Nygren, supra, and allow use of the devices incommercially viable and competitive manner. Indeed, the sensitivity ofthe devices so far surpasses existing techniques that existent "gold"standards of detection are exceeded by devices and methods of thisinvention.

An "optically active surface" is a surface that participates in thegeneration of an optical effect such that the light impinging upon thatsurface is in some way altered. Such optically active surfaces may beadapted to respond not only to polychromatic light (e.g., white light)but also to monochromatic light (e.g., laser light, which may beinherently polarized). Devices of this invention preferably produce acolor signal that strongly contrasts the background interference colorof the unreacted test surface and a reacted surface. The test surfacemay produce various shades or intensities of color that correspond to asemi-quantitative measurement of the analyte concentration in thesample, and may be visually or instrumentally measured. Such devicesallow the quantitative, instrumented analysis of thin film assaysystems.

In one embodiment, the optically active surface has a non-specularsurface, or is provided with a transparent layer having a non-specularsurface through which the optically active surface may be viewed. Thisembodiment is useful in the invention since it makes the angle fromwhich the surface is viewed less important. The term "non-specular" ismeant to indicate that the surface does not act mirror-like (specular),but provides a diffuse response to light. Generally, it includes anirregular surface with between 100 nm and 100 μm variations in height.The primary advantage is that a diffuse reflection allows the colorchange to be visible over a broad range of angles relative to theincident light.

In yet further embodiments, the substrate may include an interferencefilm which may be formed from silicon nitride, silicon oxides, titaniumdioxide, silicon oxynitride or cadmium sulfide and the like. This filmacts to cause incident light to undergo interference such that aspecific color is produced on the surface of the substrate. This filminteracts with other layers on the substrate to ensure that a colorchange or wavelength intensity change is observed when the analyte ispresent on the device. In more preferred embodiments, an attachmentlayer is provided which allows bonding of a receptor molecule specificfor the analyte of interest to be bound to the device. It is importantin the invention that this attachment layer allow attachment ofsufficient of the receptor material so that a signal is produced on thedevice. In other related embodiments, the device may be used in a mannerin which, once the analyte of interest is bonded to the attachmentlayer, other layers may be deposited on the device in ananalyte-specific manner to produce the color signal or a more intensecolor signal.

While it may be preferred to produce a device which can be analyzed byeye, the invention also includes those devices which can be used with anellipsometer, a comparison ellipsometer, a reflectometer, a profilometeror modified ellipsometers as described in this application, and thelike.

In other related aspects (described in more detail below), the inventionfeatures methods for use of the above devices, specific devices adaptedfor use in the invention, and methods for optimizing devices of theinvention by formation of a substrate having an optically active surfacewith varying thicknesses of each of the component layers such that theoptimal thickness of each layer can be readily determined.

Specifically, the invention features similar devices in which thesubstrate has an attachment layer formed from a chemical selected fromthe group consisting of dendrimers, star polymers, molecularself-assembling polymers, polymeric siloxanes, and film forming latexes;the substrate itself is formed from a material selected from the groupconsisting of monocrystalline silicon, a amorphous silicon on glass,amorphous silicon on plastic, a ceramic, polycrystalline silicon, andcomposites of these materials; and the substrate may have an opticalthin film formed from a material selected from the group consisting ofsilicon nitride, silicon/silicon dioxide composites, silicon oxynitride,titanium dioxide, titanates, diamond, oxides of zirconium, and siliconcarbide.

In particularly preferred embodiments, the second color is discernablein less than one hour after contact of the analyte with the device; theresponse to light is observed when the analyte is present on the surfacein any amount selected from 0.1 nM, 0.1 ng/ml, 50 fg, and 2×10³organisms having the analyte; the surface is specular, or non-specular,or a transparent layer having a non-specular surface is provided forviewing of the optically active surface; the substrate is selected fromthe group consisting of a solid support, a flexible support, a plastic,a glass, a metal and a non-metal; the substrate is light reflective orlight transmissive; the light is monochromatic light, polychromaticlight, ultraviolet light, or infrared light; the analyte is selectedfrom the group consisting of rheumatoid factor; IgE antibodies specificfor Birch pollen; carcinoembryonic antigen; streptococcus Group Aantigen; viral antigens; antigens associated with autoimmune disease,allergens, a tumor or an infectious microorganism; streptococcus Group Bantigen, HIV I or HIV II antigen; or host response (antibodies) to saidvirus; antigens specific to RSV or host response (antibodies) to thevirus; an antibody; antigen; enzyme; hormone; polysaccharide; protein;lipid; carbohydrate; drug or nucleic acid; is derived from the causativeorganisms for meningitis; Neisseria meningitides groups A, B, C, Y andW₁₃₅, Streptococcus pneumoniae, E. coli K1, Haemophilus influenza typeB; an antigen derived from microorganisms; a hapten, a drug of abuse(including drugs which are unlawful to use without a permit or license);a therapeutic drug; an environmental agents; and antigens specific toHepatitis; the non-specular surface has a reading of between 2700 and3295 with a profilometer, wherein this value represents the RMSroughness divided by the average peak height of the surface texture, andwhose specular reflectances measured by an HeNe laser light source isless than about 5%; the substrate is selected from the group consistingof glass, and plastic, comprising a layer of amorphous silicon on itssurface, whereby an optically active surface is produced; the opticallyactive surface includes monocrystalline silicon or metal; the substrateis metal further having a layer of amorphous silicon; a receptor layerreceptive to analyte is provided with a; specific binding partner forthe analyte; the receptor layer is formed from material selected fromthe group consisting of antigens, antibodies, oligonucleotides,chelators, enzymes, bacteria, bacterial pili, bacterial flagellarmaterials, nucleic acids, polysaccharides, lipids, proteins,carbohydrates, metals, viruses, hormones and receptors for saidmaterials; and the first color is golden in appearance and the secondcolor is purple or blue in appearance to the eye.

In other preferred embodiments, the device is configured and arranged toprovide a symbol detectable by eye in response to polychromatic light;and the optical film is coated on the device in a thickness between 480Å and 520 Å; and the analyte of interest is sandwiched between thereceptive material and a secondary binding reagent.

In another aspect, the invention features a device for use in an opticalassay for an analyte, which includes a multi-layered substrate formedwith a layer of base material, a conducting metal layer of aluminum,chromium, or a transparent conducting oxide, and a layer of amorphoussilicon, wherein the metal layer is positioned adjacent the amorphoussilicon. Alternatively, the device has a multi-layered substrate with alayer of base material (any solid material on which optically activelayers may be applied), and a layer of amorphous silicon adjacent thebase material. In preferred embodiments, the device has ananti-reflective layer attached to the upper substrate surface, having anoptical material able to attach to the upper substrate surface, and areceptive material positioned most remote from the upper substratesurface and selected from materials specific to bind the analyte ofinterest in a fluid to be tested; the base material is selected from anyof the group consisting of glass, fused silica, plastics,semiconductors, ceramics, and metals, and may be either rigid orflexible; and an attachment layer is interposed between the opticalmaterial and the receptive material.

In yet other aspects, the invention features an optical assay device fordetection of an analyte formed with a substrate selected from glass,plastic, silicon and amorphous silicon, an anti-reflective layerselected from silicon nitride, composite of silicon/silicon dioxide,titanates, silicon carbide, diamond, cadmium sulfide, and titaniumdioxide, an attachment layer selected from a polymeric silane, polymericsiloxanes, film forming latex, or a dendrimer, and a specific bindinglayer for the analyte.

In preferred embodiments, the amorphous silicon layer has a thicknessbetween about 900 and 1100 nm; an aluminum layer of between about 1800and 2200 Å thickness is provided on the glass; the silicon nitride,composites of silicon/silicon dioxide, titanates, or titanium dioxidelayer has a thickness between about 480 and 515 Å; the attachment layeris an aminoalkyl-T-structured branched siloxane of between about 90 and110 Å thickness; and the receptive material is an antibody layer ofbetween about 30 and 60 Å thickness.

In more preferred embodiments, the substrate is configured and arrangedso that any change from the first color to the second color is indicatedby the output of an instrument, such as an ellipsometer; the change isin the intensity of light reflected or transmitted from the surface; theimpinging light is reflected by the device and the reflected light iselliptically or linearly polarized, monochromatic, polychromatic,unpolarized, visible, UV, or IR, or any combination thereof; thesubstrate supports an optically active surface or is optically activeitself.

In another aspect, the invention features a method for detecting thepresence or amount of an analyte of interest in a sample, including thesteps of providing a device as described above, and contacting theoptically active surface with a sample potentially including the analyteof interest under conditions in which the analyte can interact with theoptically active surface to cause the optically active surface toexhibit the second color when the analyte is present. An optical readermay be used to measure the change in the second color. An optical readerconsists of one of the following group of instruments: an ellipsometer,a refletomer, a comparison ellipsometer, a profilometer, a thin filmanalyzer, or modifications thereof.

In preferred embodiments, the analyte of interest is sandwiched betweena receptive material (e.g., an antibody or antigen) and a secondarybinding reagent (e.g., an antibody or antigen); the analyte of interestis detected directly by the binding of the analyte; the analyte ofinterest is detected by competition with a signal generating reagent forthe receptive material; the analyte of interest is detected by indirectsignal generation; the sample is selected from the group consisting ofurine, serum, plasma, spinal fluid, sputum, whole blood, saliva,uro-genital secretions, fecal extracts, pericardial, gastric,peritoneal, pleural washes, vaginal secretions, and a throat swab; andthe method includes using a reflectometer to measure the change in coloror intensity.

In particularly preferred embodiments, the method involves contactingthe substrate with a test sample potentially containing the analyteunder conditions in which the substrate exhibits the second color whenthe substrate includes the analyte in the above amount in less than onehour; and the device is a reflectometer set such that the first color isa background intensity of a specific wavelength or range of wavelengthsof light, and the second color is a change in intensity of one or moreof those wavelengths of light relative to the first color; or the deviceis a thin film analyzer set such that the first color is a backgroundintensity of light transmitted through the analyzer to a detector, andthe second color is a change in intensity of the light transmittedthrough the analyzer to the detector relative to the first color; or thedevice is set such that the first color is an eye-observableinterference color, and the second color is a change in color relativeto the first color.

In yet another aspect, the invention features a method for producing anoptical assay device having a substrate and one or more optical layers,an attachment layer and a receptive layer, by spin coating one or moreof the layers.

In preferred embodiments, the method involves spin coating an opticalthin film on the surface of a substrate, where the film is formed fromone or more of the group consisting of: polysilizanes, aluminumalkyloxides, silicates, titanates, zirconates, and T-resin siloxanes,and the film has a thickness between 250 and 550 Å; the method includesspin coating an attachment layer on the optical device on the opticalsurface of the device, most preferably formed from one or more of thegroup consisting of: non-linear branched polymeric siloxanes, filmforming latexes, and dendrimers, with a thickness between 25 and 250 Å;and the receptive material is spin coated or solution coated to theattachment layer.

In another aspect, the invention features an optical assay device havingan active receptive surface supported on a pedestal and held within afirst container; the first container includes a first absorbent materiallocated at the base of the pedestal, configured and arranged to absorbliquid draining from the surface, a second container, hingedly connectedto one side of the first container, the second container having a secondabsorbent material, wherein the second container can be closed to thefirst container by rotation about the hinge, and wherein such closingcauses the second absorbent material to contact the surface.

In preferred embodiments, the second container further has a handleconfigured and arranged to cause the second absorbent material to moverelative to the location at which the second absorbent material contactsthe surface; the device further has a movable flap in the secondcontainer which is configured and arranged to prevent the secondabsorbent material from moving from the second container; the device hasa movable flap in the first container which is configured and arrangedto prevent the second absorbent material from moving from the firstcontainer; each flap is hingedly connected to the first or secondcontainer, and is provided with one or more apertures to allow access tothe surface or the second absorbent material.

In a related aspect, the invention features an optical assay device witha plurality of optically active surfaces supported on a base, the basehaving a first absorbent material configured and arranged to absorbliquid draining from the surfaces, and a slidable lid having one or moreabsorbent regions configured and arranged to contact the opticallyactive surfaces during use of the device.

In preferred embodiments, the device is provided with step means toallow stepped movement of the lid relative to the base; the lid has aseries of apertures which allow selected access to the surfaces duringuse of the device; the lid has an elongated aperture and wherein thebase comprises a series of indicia, wherein the elongated aperturecooperates with the indicia to indicate a method for use of the device;the analyte of interest is the Human Immunodeficiency Virus (HIV) I orII or a combination thereof, Streptococcus Group A, Streptococcus GroupB, RSV (Respiratory Syncytial virus), Hepatitis B, a Chlamydia species,HSV (Herpes Simplex virus), an antigen, an antibody, nucleic acid,oligonucleotides, chelators, enzymes, bacteria, viruses, hormones andreceptors for the materials; and the device is configured and arrangedto measure the presence of amount of Streptococcus A antigen,Streptococcus B, RSV, Chlamydia or a Hepatitis antigen; and has anoptically active receptive surface.

In another aspect, the invention features an optical assay device,having an optically active receptive surface configured and arranged toallow simultaneous assay of a plurality of samples on the surface forone analyte of interest, and an automated liquid handling apparatus(e.g., a pipetting device) configured and arranged to dispense sampleand reagent solutions to the surface.

In preferred embodiments, the device further has an optical reader todetermine the result of each assay; the device has a blotting or blowingmeans configured and arranged for drying the surface; and the deviceprovides a quantitative or qualitative assessment of a sample applied tothe device.

In another aspect, the invention features a method for detecting ananalyte of interest, by the steps of providing a detection device havinga light reflective or transmissive substrate supporting one or morelayers with an adhering attachment layer to which is affixed a receptivematerial which specifically interacts with the analyte of interest,reacting the device with a sample potentially containing the analyteunder conditions in which the analyte binds to the receptive material,and reacting bound analyte with a reagent which creates a mass change onthe surface of the device.

In preferred embodiments, the device has a substrate of planarreflective material supporting an attached layer of immunologicallyactive material; the substrate consists of a planar reflective material;the substrate and the attached layer polarize radiationellipsometrically upon reflection; the reagent increases or decreasesthe mass on the device, e.g., the reagent is an enzyme, or includes apolymeric latex, such as a film forming styrene-butadiene copolymerwhich is covalently attached to a secondary receptive material specificto the analyte of interest; most preferably, the reagent is an enzymeconjugate, which includes an anti-bacterial-antibody-enzyme complex; thereagent causes precipitation of mass by a precipitating agent, such as asubstrate for an enzyme, e.g., containing3,3',5,5'-tetramethylbenzidene.

In a related aspect, the invention features a kit for an optical assayfor an analyte of interest having a test device with an optically activesurface reactive with the analyte, and a reagent adapted to react withthe analyte bound to the surface to alter the mass on the surface.Preferably, the reagent is an enzyme conjugate or a polymeric latex.

In another related aspect, the invention features a method for detectingan analyte of interest in a sample, by the steps of providing a thinfilm optical immunoassay device having a substrate, having an upper anda lower surface, and supporting on its upper surface, an unlabeledantibody layer bound to the substrate, at least one layer containing theanalyte from the sample, the analyte containing layer supporting atleast one layer having an enzyme conjugate complexed with the analyte;contacting the enzyme conjugate with a precipitating agent; incubatingfor a time period sufficient to cause precipitation of product frominteraction of the precipitating agent and the enzyme; and opticallymeasuring the mass change of the enzyme conjugate layer and theunlabeled antibody layer as an indication of the amount of the analytein the test sample.

Preferably, the enzyme conjugate has an immobilized peroxidase or ananti-bacterial anti-body-horseradish peroxidase complex; or the enzymeconjugate is alkaline phosphatase and comprises ananti-bacterial-antibody-alkaline phosphatase complex; and theprecipitating agent is a substrate containing 5-bromo-4-chloro-3-indolylphosphate.

In another aspect, the invention features an instrument configured andarranged to detect the presence or amount of an analyte of interest onthe substrate of an optical device. The instrument has a source oflinearly polarized, monochromatic light positioned at an angle otherthan Brewster's angle relative to the substrate, and an analyzerpositioned at the angle relative to the substrate at a location suitablefor detecting reflected polarized light from the substrate. The analyzeris configured and arranged to approximately maximize change in theintensity of the light reflected from the substrate that is transmittedthrough the analyzer when a change in mass occurs at the substraterelative to an unreacted surface.

In another aspect, the invention features a method for optimizing anoptical assay device for an analyte, by the steps of providing-asubstrate having a chosen thickness of an optically active layerthereon, providing an attachment layer of a chosen thickness on theoptical coating, providing a receptive layer of a chosen thickness forthe analyte, wherein at least one of the thicknesses of the opticallyactive layer, attachment layer and receptive layer is varied to providea plurality of thickness of the layer, contacting analyte with thereceptive layer under conditions in which an increase in mass on thereceptive layer results, and determining the optical thickness of the atleast one thickness of a the layer. Preferably, the thickness of opticalcoating is varied incrementally along the length of the substrate.

Applicant has discovered that one feature useful for optimization of theclaimed devices is the use of a substrate having a known refractiveindex having an anti-reflective layer attached to the upper substratesurface consisting of at least one layer of a material able to attach tothe upper substrate surface and at least one layer of a receptivematerial positioned most remote from the upper surface and selected frommaterials specific to bind said analyte of interest in a fluid to betested. It is important that the anti-reflective layer has a refractiveindex which is approximately the square root of the known refractiveindex of the substrate surface material adjacent to the anti-reflectivelayer, and has a thickness less than an odd number multiple of aquarterwave of the wavelength of a light incident upon the device.

That is, applicant has discovered that in order to optimize devices ofthe present invention it is necessary to deviate from previouslydeveloped mathematical algorithms (see Table 3, of the "Handbook ofOptics", Walter G. Driscoll, and William Vaughan, editors, McGraw-HillBook Co., New York, 1978, pp. 8-48 to 8-49) reflecting the desiredthickness of specific optical layers in the device (see Background ofthe Invention). Thus, while such mathematical formulae may provide anindication of a general thickness that might be useful in a ratherinsensitive device, the method of this invention provides a device withsignificantly and surprisingly greater sensitivity.

Below is provided an indication of the methodology by which the optimalmaterials and methods useful for construction of optical test surfacesof this invention can be made. Generally, the present invention includesnovel optically active test surfaces for the direct detection of ananalyte, whether through colored signal generation detectable by eye orinstrumented analysis. These test surfaces have a specific receptivematerial bound to the test surface by use of an attachment layer. Thus,the present invention provides a detection method which includesselecting an optical substrate, attaching receptive material specific tothe analyte of interest on the upper layer of the substrate, contactingthe receptive material with a sample fluid containing the analyte ofinterest, and then examining the change in reflection or transmissionproduced at the coated surface by observing a change in first color.

The present invention has a broad range of applications and, may beutilized in a variety of specific binding pair assay methods. Forinstance, the devices of this invention can be used in immunoassaymethods for either antigen or antibody detection. The devices may beadapted for use in direct, indirect, or competitive detection schemes,for determination of enzymatic activity, and for detection of smallorganic molecules (e.g., drugs of abuse, therapeutic drugs,environmental agents), as well as detection of nucleic acids.

Devices of this invention feature a test surface suitable to performingassays which can be developed from a wide variety of substrates,anti-reflective, attachment, and receptive materials which can beintroduced into a user friendly, broadly applicable assay device andprotocol, may be used in a format which allows multiple test resultsfrom a single assay, and may be used to allow multiple analytes to betested with a single sample in a simple way.

The use of an optical thin film or anti-reflective (AR) coating is onecomponent in the device responsible for the observed color change. Thedevices of the present invention also provide improved performanceattributes for assays which do not require this level of sensitivity.The improved performance attributes include length of assay, ease ofinterpretation, and flexibility in assay format and protocol.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings will first briefly be described.

Drawings

FIGS. 1A (unreacted test surface) and 1B (reacted test surface) arediagrammatic representations of the interference phenomena central tothe devices and methods of the present invention;

FIGS. 2A and 2B are diagrammatic representations of specular (FIG. 2A)and non-specular or diffuse (FIG. 2B) substrate surfaces;

FIGS. 3A-G are a diagrammatic representations of a method of the presentinvention for selection of optimal interference films for use in devicesof the present invention;

FIG. 4 is a representation of the attachment of3-aminopropyltriethoxysilane to an optical surface;

FIG. 5 is a diagrammatic representation of the attachment of multivalentsiloxanes which are useful to produce devices of the present invention;R may be any one of a number of groups which do not interfere with theattachment of the chemically active groups to the silica atoms presenton an optical surface, and which do not interfere with the laterattachment of receptor (e.g., biological) moieties, for example, such Rgroups may be primary, secondary or tertiary amines, alcohols, ethoxygroups, phenyl groups and aromatic groups, among others;

FIGS. 6A-F are a diagrammatic cross-sectional representations of devicesof the present invention, which are useful for either instrument readresults (FIGS. 6A-C), or for eye-visible readable results (FIGS. 6D-F).Materials and layers are designated as follows: substrate (1), opticalthin film (2), attachment layer (3), receptive material (4), amorphoussilicon (5), metal film (6), and composite interference film (7);

FIGS. 7A-E are diagrammatic representations of methods of the presentinvention in which an optical signal is obtained or enhanced byprovision of mass on the optical surface; e.g., by use of an antibodyhaving a latex bead (FIGS. 7A and 7B), or by use of an enzyme labeledantibody to cause catalytic deposition of product material onto asubstrate (FIGS. 7C-D). Materials and layers are designated as follows:substrate (1), optical thin film--optional (2), attachment layer (3),receptive material (4), amorphous silicon (5), metal film (6), analyte(7), and mass enhancement (8);

FIGS. 8A-8G are isometric and exploded views of a device of the presentinvention; specifically, FIGS. 8A, 8B, 8C, and 8D are respectively a topview, a bottom view, an isometric side view, and a side view of thedevice, FIG. 8E is an isometric view of the device opened for use in anassay, FIG. 8F is an exploded view of the test surface of the device,and FIG. 8G is an exploded view showing various components within thedevice;

FIGS. 9A-9E are isometric and other views of a multi-test device of thepresent invention, specifically, FIGS. 9A, 9B and 9C are respectively atop view, an isometric view, and an exploded view of the device, FIG. 9Dshows the back of the front cover of the device, and FIG. 9E is the topof the device without the front cover;

FIGS. 10A-I are a diagrammatic representation showing method steps foruse of the device shown in FIGS. 8A-G;

FIGS. 11A-E are a diagrammatic representation showing a method of use ofa device similar to that shown in FIGS. 9A-E;

FIGS. 12A-C are a diagrammatic representation of a potential batchsampling formats;

FIG. 13 is a diagrammatic representation of a prior art ellipsometeroptical path;

FIG. 14a is a diagrammatic representation of a thin film analyzer usefulin the present invention which uses a monochromatic light source and asingle photodiode or array;

FIG. 14b is a diagrammatic representation of a polychromatic lightsource and a photomultiplier detector;

FIG. 15 is a diagrammatic representation of a modification of the priorart ellipsometer's optical path which demonstrates a new method forsignal detection; and

FIG. 16 is a diagrammatic representation of an ellipsometer having areduced optical pathway length; and

FIGS. 17A-C are a diagrammatic representation of the advantages of areflective substrate in fluorescent assay methods relative toconventional fluorescent methods;

FIG. 18 is a diagrammatic representation of a fifth generation starpolymer or dendrimer (molecular self-assembling polymer).

TEST DEVICE

A number of types of optical thin film monitoring technology includingellipsometry, multiple angle reflectometry, interference spectroscopy,profilometry, surface plasmon resonance, evanescent wave, and variousother forms or combinations of polarimetry, reflectometry, spectroscopy,and spectrophotometry are useful in this invention. This inventionconcerns the application of such technologies for the detection ormeasurement of changes in the thickness, density, or mass of thin filmsresulting from the concentration-dependent immobilization of analytes ona surface of suitably selected binding material. Such thin film assaytechnologies directly detect or quantitate the material of interest, andare alternatives to conventional solid phase assays. Thin filmengineering problems have obstructed the development of test kitssuitable to compete with existing diagnostic or other assay-markets.

There are several critical features to the construction of a testsurface of a device of this invention which combines specific bindinglayers and optical materials. More specifically, special considerationsare required for combination of specific binding material with ananti-reflective or interference film. Each feature is discussed below,but in general, one must evaluate the interrelationships between anoptical substrate, an optional optical thin film or interference film oranti-reflective (AR) film, an attachment layer, and the receptivematerial to be used in the composite test surface, as well as the finalassay device requirements. The desired end use, visual/qualitative,instrumented/quantitative, and instrumented/qualitative will determinewhich feature of each component is selected in the production of a finaluseful test device of appropriate sensitivity and performancecharacteristics.

Referring to FIGS. 1A and 1B, there is shown the general phenomenon oflight interference that is central to the utility of one embodiment ofthe present invention. This phenomenon is generally independent of themacroscopic surface characteristics of the test device. For example, itis important only that the device cause a change in the observed colorof light reflected from the surface, and it is not necessary to provideany specific pattern on the surface, such as a diffraction grating orany related pattern. Thus, generally the surface is a planar surfacewith no specific pattern provided thereon. However, the surface may beprovided in a shape or design that is visually useful to the human eye.An unreacted test surface causes white light incident at the device tobe reflected as gold light, whereas a reacted test surface, due to theadditional matter from analyte binding will cause the incident whitelight to be reflected as purple or blue light. The change from gold topurple or blue indicates the interference difference between the reactedand the unreacted test surfaces.

FIGS. 6A-F show in a diagrammatic form the general structure of varioustypes of test surfaces of devices utilizing the present invention. Foran instrument-read device the surface is provided with a substrate, anattachment layer and a receptive material layer, and may optionally alsobe provided with amorphous silicon and/or a metal film. In contrast, forvisually readable devices it is necessary to provide an optical thinfilm (or an interference film) which, together with the attachment layerand receptive material layer, form a composite interference film. Thesevarious layers and their interactions are now discussed by way ofexemplification.

Substrate

One or more thin films on a surface may attenuate incident light on thatsurface producing a change in the incident light that may be measuredeither by reflectance or transmittance. Reflection occurs when lightencounters a medium of a different refractive index than the ambientmedium. The ambient medium is generally air with a refractive index of1.0. Transmission is a general term describing the process by whichincident light leaves a surface or medium on a side other than theincident surface. The transmittance of a medium is the ratio of thetransmitted light to that of the incident light. Both the reflected ortransmitted light can be detected by eye, or may be measured withinstrumentation. In this invention, use of such light attenuation in anyspecific device as a measure of the amount of an analyte in a sample ispossible. The actual structure of the chosen device, however, depends onwhether a reflection or transmission mode is desired, and whether theresult is to be interpreted by eye or with an instrument. These specificcombinations are relevant to the choice of a substrate(s) and aredescribed generally below.

Reflectance Mode, Interpretation By Eye

One example of the phenomenon used in this invention is the interferencecolors observed when viewing oil on water on an asphalt surface. Thisinterference effect is very common, and can be seen in a piece ofmultilayered mica, a fragment of ice, a stretched plastic bag, or a soapfilm. The change in color is due to local variations in the thickness ofthe material. The colors observed with oil on water are particularlyintense and easily observed by the eye due to the difference inrefractive index between water and oil. The colors are furtherintensified because the water provides a mirror-like (specular)reflection. The asphalt surface serves to absorb transmitted light,suppressing back reflection, which would tend to dilute the colorsobserved. The eye is more sensitive to contrast than to changes inintensity, therefore, selection of materials should allow for theproduction of colors which provide high contrast as a result of mass orthickness change at the surface. Films may be added to the surface of amaterial to modify the reflectance of one or more wavelengths or band ofwavelengths. These types of materials are used to produce sunglasses,camera lenses, and solar windowpanes.

When the test surface is designed to produce a color change visible tothe eye, the optical substrate must provide a surface that is reflectiveonly at its uppermost surface, and of a known refractive index.Polished, monocrystalline silicon, metals, and some ceramics or darkglasses provide surfaces which may be used directly in this application.These materials inherently contribute to the generation of the observedsignal and may be considered to be optically active.

Materials such as glass or plastics may require additional processingbefore they are useful in this technique. A material such as glass willallow reflection to occur at its upper and back surfaces. To preventthis, and allow such materials to be utilized, an additional film mustbe applied to the uppermost surface. Amorphous silicon, a thin metalfilm, or a combination of these materials may be used. In this case theglass serves as a solid support and is not inherently involved in thegeneration of the observed color, therefore it is considered to beoptically passive.

Only the refractive index of the uppermost surface is important in theselection of the optical thin film or anti-reflective (AR) coating wheneither a single substrate material or a more complex structure isused(see below). AR materials compatible with a preselected substrateshould approximate the calculations given in Table 3 pp. 8-48 to 8-49 ofthe "Handbook of Optics". With monocrystalline silicon this is simplythe uppermost surface, with transparent glass this is the surface coatedwith amorphous silicon or other material. Adjustments to the AR filmthickness are made using the wedge experiment described below. Using areflective substrate to produce a color change perceived by the eye, theaddition of a film of suitable refractive index and thickness is anabsolute requirement in determining which wavelengths of light areantireflected.

The optical substrate materials may produce a specular reflection, ormay be treated to, or intrinsically produce, a diffuse reflection whichis less angle dependent in viewing the signal, as discussed below.

Transmission Mode, Interpretation By Eye

For this technique, the color produce is not viewed in the reflectedlight, but is observed as the light is transmitted through a surface.This selective transmission of different wavelengths of light is used toproduce sunglasses, camera lenses, windowpanes, and narrowbandpassfilters. The materials will selectively reflect and transmit differentwavelengths of light. A narrowbandpass filter will reflect a large bandof wavelengths of light, and will selectively transmit only a very smallband of wavelengths centered around one specific wavelength. Thenarrowbandpass filter is constructed of an optical glass which is coatedon one side with a material which will reflect many wavelengths oflight. A change in the thickness of the material which coats the opticalglass will change the useful range of the filter centering on a new setof wavelengths.

For this application, the optical substrate selected must betransmissive to the visible wavelengths of light, thus materials such asmonocrystalline silicon, metals, certain plastics and ceramics are notsuitable unless they are extremely thin, transparent sections. Glassesand certain transparent plastics are the most useful for thisapplication. In this type of technique the substrate is opticallyactive. For the generation of a color change visible to the eye, therefractive index of the substrate impacts the type of AR film which isselected. A uniform or smooth surface is required for use in thisapplication to prevent loss of signal due to scattering at one or moreof the transmitting surfaces.

A glass substrate coated with a layer of amorphous silicon may betransmissive to visible light at certain angles, if the amorphoussilicon layer is sufficiently thin. This is also true for a very thinlayer of metal on a glass substrate. For this type of test surface, theviewing should be arranged such that the amorphous silicon is the backsurface of the test piece (i.e., opposite to the viewing surface).

Reflectance Mode, Instrumented Interpretation

The use of the AR or optical thin film component is optional when aninstrumented detection is utilized. A reflectometer requires a colorchange or change in luminosity (intensity) for generation of a signal.This color change may be different from the color change selected forvisualization by eye, as the instrument will record changes in intensityand does not require a maximal change in contrast. AR film thickness ispreferably adjusted to provide the maximal change in recorded intensityas a function of analyte binding. In addition, modifications to thereflectometer will allow it to also measure changes in color/luminosity(intensity) with a specularly reflecting or diffusing reflectingsurface.

For use with the ellipsometric type of instruments the optical substrateshould provide a specular reflection. Reflection should occur only fromthe uppermost surface. As previously discussed, glass serves only as asupport in this case and is optically passive or not involved in thegeneration of the detected signal. Instrumented detection will observe achange in light intensity due to its interaction with the thin films.The light may be elliptically or linearly polarized, polychromatic,monochromatic, and of any wavelength desired.

Transmission Mode, Instrumented Interpretation

Any optical substrate which is transparent to the incident light may beused in this application, whether that light is polychromatic,monochromatic, linearly polarized, or elliptically polarized, and of anywavelength desired. Use of an AR film is optional in this application,but if required for use with a reflectometer, the rules presented forthe interpretation by eye also apply here. Thus, the refractive index ofthe optical substrate influences the selection of the AR coating. Designof the reflectometer is easily modified to allow reflection ortransmission measurements to be made.

When a change in the transmitted light is to be made independent of anycolor, an AR film is not required. The only requirement for thesubstrate in this application is that some component or components ofthe incident light be transmitted, and that a change in mass orcharacter on the uppermost surface of the test piece modifies thetransmitted light in a detectable manner. Materials such as the Irtranseries produced by Eastman Kodak may be of use in this application formonitoring changes in the infrared (IR) properties of these films.

Thus, the term "substrate" includes not only a solid surface for holdingthe layers described below, but also an optically active substrate whichmay include an optical thin film. For clarity, these two portions of asubstrate are discussed separately, but those in the art will recognizethat all that is essential in this invention is that the layers (towhich the attachment layer and other layers are attached), be opticallyactive to provide a detectable change in the thickness or mass of theselayers as described above.

The optical substrate is either a solid material, or supports a layer ofmaterial which acts optically. These materials must have a knownrefractive index if it is to be combined with an optical thin film toproduce an interference effect. Thus, it may be formed from any desiredmaterial which is reflective or made reflective, as discussed below. Forinstrument use, the substrate can also be transparent (e.g., glass orplastic) so that transmitted light is analyzed.

This invention is suited to use of a variety of optical substratematerials and formats to suit the needs of the end user. The opticalsubstrate can be formed of, or have coated on it, a material thatprovides either diffuse or specular reflection, it may be rigid orflexible, reflective or transmissive, and it may form an opticallyfunctional component of the test surface, or it may act as an opticallypassive support (and be provided with optically active layers). Devicesdesigned for instrumented analysis may not require an anti-reflective(optical thin film) coating on the substrate, while those designed forviewing by eye may require such a coating. Criteria useful for selectingan optical substrate for instrumented applications, or for viewing byeye of a color-signal generating application, are presented below.

A wide range of rigid materials may form the optical substrate,including glass, fused silica, plastic, ceramic, metal, andsemiconductor materials. The substrate may be of any thickness desired.Flexible optical substrates include thin sheets of plastic and likematerials. Most substrates require only a standard solvent, plasmaetching, or acid cleaning, well known to those skilled in the art,before subsequent layers may be deposited on them.

For color-signal generation visible to the eye, an anti-reflectivecoating material is required. Polymer films, such as mylar (polyethyleneterapthalate) and other materials having a low surface energy may notadhere well to such material and may require additional treatment beforethis layer can be deposited. To improve adhesion these opticalsubstrates may be etched in an oxygen plasma, under conditions standardfor oxygen plasma cleaning in semiconductor processing.

The surfaces of many solid materials, such as glass, and semiconductormaterials, such as silicon, metals, etc., are sufficiently smooth toprovide specular reflection if they are polished. For use in areflection-based assay the major requirement in selecting an opticalsubstrate is that the reflection occur, or be made to occur, only at theupper surface. This is especially critical for devices which include aninterference film and are to be viewed by eye. This is easilyaccomplished by vapor deposition of a thin metal film on the substrate,and attachment of subsequent layers by techniques known to those skilledin the art. For example, the uppermost surface of a glass substrate maybe coated with a layer to prevent unwanted reflections from the lowersurface.

Metal Layer

If the substrate is to be used in a reflection mode, and is partially orfully transparent, it may be coated with an opaque material to blocktransmitted light and allow reflection to occur only from the uppersurface. For example, a glass substrate may be coated with a layer ofaluminum, chromium, or other transparent conducting oxide, by mountingin a vacuum chamber facing an aluminum-filled tungsten boat. The chamberis evacuated to a pressure of 1×10⁻⁵ Torr. Current is passed through thetungsten boat, raising it to a temperature at which the aluminumdeposits on the substrate at a rate of 20 Å/second for 100 seconds,coating the glass with an opaque layer of aluminum having a thickness of2000 Å. Thinner layers of aluminum or chromium may also be used toeliminate any back surface reflections. Non-conducting depositiontechniques may be used to deposit the metal film.

Amorphous Silicon

The aluminum-coated glass, described above, may be considered opticallypassive. Thus, if it is coated with a layer of hydrogenated amorphoussilicon (a-Si:H), the optical characteristics of the substrate will bederived from the a-Si:H alone. The aluminum-coated glass is requiredonly when the amorphous silicon deposition process requires a conductingsurface. Techniques which do not require the use of a conducting surfacefor the deposition of amorphous silicon are known. To produce thissubstrate, the aluminum-coated glass is mounted on one of two opposingelectrodes in plasma-enhanced chemical vapor deposition system. Thesystem is evacuated, and the substrates are heated to 250° C. A constantflow of silane (SiH₄) gas into the chamber raises the pressure to 0.5Torr. A plasma is struck by applying 10 mW/cm² of RF power to theelectrodes. A film of a-Si:H deposits on the substrates, and grows to athickness of approximately 1000 nm in about 75 minutes. The a-Si:H soformed may form the first optically functional layer on the testsurface.

A glass substrate coated only with a-Si:H (without the aluminum layer)is also useful in this invention. Transparent substrates, such as glass,fused silica, sapphire, and many plastics may be used in instrumenttransmission measurements, without additional modification. Color-signalgeneration visible to the eye is possible with a transmissive substratewhere the anti-reflection properties of the coatings are determined fromthe transmitted light.

Many of the substrates with a sufficiently reflective surface for thinfilm measurements are formed of metals. Examples of these metals,include but are not limited to, iron, stainless steel, nickel, cobalt,zinc, gold, copper, aluminum, silver, titanium, etc. and alloys thereof.Metals are particularly useful substrates when an instrument detectionmethod is employed. For instrumented measurement systems, the mainrequirement is that the substrate be reflective and planar. In contrast,for eye visible color signal generation it is very difficult, but notimpossible, to match the reflectivity of the metal with a suitableanti-reflective coating. The reflectivity of the optical substrate andthe optical thin film (see below) used must match for the optimalproduction of an interference color. Thus, devices designed for colorproduction are generally formed from other substrates, or from amorphoussilicon-coated metal substrates as discussed above.

Non-Specular Surface

Referring to FIG. 2A, there is a diagrammatic representation of ageneral concept of the present invention in which rather than providinga specular substrate (in which the surface is mirror-like or almostmirror-like), the surface is manufactured in a manner which will createirregular bumps as shown diagrammatically in FIG. 2B and referenced bythe symbol B. These bumps are significantly exaggerated in the figureand are generally of a size between 1 nm and 100 μm, most preferablybetween about 100 nm and 100 μm. Again, these bumps are not provided inany regular manner, (such as in the form of a diffraction grating), butrather are provided simply to cause a general scattering of lightincident upon the surface. By such provision it becomes unimportant fromwhich direction the light incident on the substrate is angled and thecolor change noted in FIG. 1B can be observed by holding the substrateat any angle to incident light, or to the observing eye. Both instrumentand eye-visible color signal generation surfaces can be constructed withspecular or diffuse reflecting substrates.

A surface that provides a diffuse reflection can be obtained in severalways: physical abrasion, chemical abrasion, or coating of a material.Specular substrates may be roughened by physical abrasion using acompound containing grains of silicon carbide to form a diffuse surface.Alternatively, the material may be chemically abraded. For example, amonocrystalline silicon wafer may be etched in an aqueous solution of30% potassium hydroxide (by weight) at 80° C. to form a rough surfacecomposed of pyramid structures. The abrasion process may be followed byan isotropic etch, well known to those skilled in the art, to yield anirregular surface that produces a diffuse reflection.

The diffusing properties of the substrate may also be produced by acoating. For example, polystyrene spheres having a diameter of 2 micronsmay be suspended in a fluid, such as a polyamide-containing solution. Aglass slide is vacuum mounted onto a spin coater, and the centralportion of the slide covered with the solution. The spin coater isswitched on for several seconds at 3000 rpm, causing the spheres in thesolution to disperse uniformly over the surface. The fluid is allowed todry, yielding a surface that produces a diffuse reflection.

Embodiments of the present invention include use of an optical substratewith an irregular surface to produce diffuse light reflection. They alsoinclude use of a smooth optical substrate surface covered with, overlaidby, or observed through, a light diffusing or light modifying materialsuch as or textured plastic. Viewing through such a plastic produces asimilar effect to that discussed above.

In one example, the optical substrate is formed from a silicon crystalwhich is grown and extruded to 4 inches in diameter and then diamondsawed to form a wafer. The wafers are treated with chemical etchants tosmooth the surface and reduce flaws. The wafers are lapped or groundwith aluminum oxide, titanium oxide, or silicon carbide particles in atalc slurry. The initial grain size is large and successively smallerparticle sizes are used to produce an increasingly smoother surface.Both sides of the wafer are subjected to this process. The final lappingprocess leaves a very diffusely reflective surface. Wafers may befurther processed with chemical or plasma etching to modify the diffusereflecting characteristic of the substrate.

Once the wafers are lapped, they are cleaned using the following processor a known modification thereof: the wafers are sonically cleaned with acationic detergent, followed by a rinse with 18 megaohm water. Then theyare cleaned with an anionic detergent, followed by a rinse in 18 megaohmwater. They are ultrasonically cleaned with an aqueous ammonia solutionmade of 370 ml of 30% H₂ O₂, 250 ml of aqueous ammonia and 9 gallons ofwater, and are rinsed in a cascade of water with the final rinse beingwith 0.1 micron filtered water. They are then spin-dried and are readyfor optical coating. An alternative to this procedure is the "RCA Clean"described in Polymer Surfaces and Interfaces, edited by W. J. Feast andH. S. Munro, John Wiley and Sons, New York, N.Y., page 212, 1987.

For glass, the degree of surface character or the irregularity isdiscussed in terms of gloss. The diffuse reflective capability of thesurface described here refers to the degree to which the reflection isscattered compared to a pure specular reflection. Diffuseness is afunction of the surface topography and because the relevant topographyis much larger than the interference film or biofilms, the fuzziness isnot expected to vary significantly for different specific bindingmaterial. For eye-visible color-signal generation the film will affectthe lightness or color of the reflected light, but not its diffusecharacter. Diffuse surfaces which produce color signal are particularlyuseful with reflectometers.

The surface topography, and hence fuzziness or irregularity may becharacterized with a surface profilometer, such as the Dek-tak® (SloanTechnology Corp., Santa Barbara, Calif.). The Dek-tak® provides readingson the separation or distance between surface features and an averagevalue for the height of surface features over a defined region of asurface. One useful measure of the surface is the Root Mean Square (RMS)or average surface roughness divided by the average peak spacing, wherea peak is defined to be a protrusion with a height of at least 50% ofthe RMS roughness. Since roughness is a function of the reflectivityversus angle, it may be quantified by measuring the angle dependence ofthe reflectivity. For a light source incident at 30° from normal, thereflected light intensity on a photodiode should be measured as afunction of the angle from 0° to 90°. The wafer selected shouldoptimally show a smoothly varying reflectivity over the angular rangeviewed. Using a HeNe laser light source, the specular reflectance from aroughened surface useful in this invention should be less than 5%,assuming a polished wafer reflects at 100%.

In an embodiment of the present invention, articles having thenon-specular, i.e., irregular, surfaces are characterized by peak valuesbetween about 2700 and 3295 with a preferred measurement ofapproximately 2995. This value represents the RMS roughness divided bythe average peak of the textures.

In addition to abrasive lapping, a wide range of chemical or plasmaetching technique are suitable for providing the diffuse properties ofthe substrate. For example, glass can be modified to a diffuse lightreflecting surface using a HF etch as in the production of frostedglass.

For the color-signal generation, substrate selection will determine thecharacteristics of the anti-reflective material or materials used insubsequent coating steps. Below is described the selection ofanti-reflective materials based on initial substrate selection.

The substrate material may be cut, sawed, scribed, laser scribed, orotherwise manipulated into the desired test piece configuration.Suitable test pieces for a single use assay are 0.5 cm² to 1 cm² with0.75 cm² being preferred. Test piece sizes are not restricted to theabove, as alternative formats may require substantially more or lessreactive test surface.

Optional Optical Thin Film Material(s)

Referring to FIGS. 1A and 1B, the simplest description of a singleoptical thin film is that the substrate is coated with a thin layer ofmaterial such that reflections from the outer surface of the film andthe outer surface of the substrate cancel each other by destructiveinterference. Two requirements exist for exact cancellation of reflectedlight waves. First the reflections must be 180° out of phase and secondthey must be of equal amplitude or intensity.

In the reflection mode, the optical thin film properties of the coatingsof a device of this invention suppress the reflection of somewavelengths of light and enhance the reflection of others. This causesthe suppressed wavelengths of incident light to enter the substrate, oran opaque coating on the substrate where they are absorbed. Most of thelight of other wavelengths, whose reflection is not suppressed, does notenter the coated substrate and is reflected, however, some componentsmay be absorbed. As the optical thickness of the coating changes, therange of wavelengths in the reflected light changes. In transmissionmode, the properties of the coatings suppress the reflection of somewavelengths of light and enhance the reflection of others, as in thereflection mode. This causes the suppressed wavelengths of the incidentlight to enter the substrate and to be transmitted. Light of otherwavelengths, whose reflection is not suppressed to as great an extent isreflected, and transmitted to a lesser extent. As the optical thicknessof the coating changes, the range of wavelengths in the transmittedlight changes.

Where eye-visible color-signal generation is required (see FIGS. 6D-F,right hand side), the final assay result may also be measured byinstrumentation. Ideally, for the production of a perfect interferencefilm using only the specific binding materials discussed below, and anoptical substrate, the substrate should have a refractive index of thesquare of the refractive index of the receptor layer (see below), i.e.,(1.5)² or 2.25, variations in this number can still provide usefuldevices of this invention as will be discussed below). The materialselected should be mechanically stable to subsequent processes,reflective, and of known refractive index. It is not always possible tomatch the optical substrate to a particular film. For example, abiological film. In these cases, an intermediate optical thin film mustbe used to compensate for the lack of a suitable optical substrate. Foreye-visible color-signal generation, the substrate material is subjectto two restrictions: first, it must adhere to the optical thin filmmaterial, and second, in the simplest case, the refractive index of thesubstrate should approximately equal the square of the refractive indexof the material directly above it or, on a more complex test surface,the refractive index of the substrate should be selected to fitgenerally one of the formulae in Table 3, pp 8-48 to 8-49, of the"Handbook of Optics". For example, use of a silicon wafer with arefractive index of approximately 4.1 allows a test surface to bedesigned with a wide variety of corresponding optical thin films oranti-reflective materials. The material should be coated to a thicknessof a quarterwave for the wavelengths to be attenuated, or variations inthe formulae given in Table 3. Those skilled in the art will realizethat various other substrate materials are equally suited for use as atest surface if they satisfy the above criteria.

The optical thin film coating is deposited onto the surface of thesubstrate by known coating techniques. For example, by sputtering or byvapor phase deposition in a vacuum chamber. Various other useful coatingtechniques are known to those skilled in the art. Materials useful asoptical thin film coatings are formed of clear material which issignificantly transmissive at the thickness utilized, and suppressessome wavelength of reflective light when coated onto the substrate. Thefilm, once deposited onto the optical substrate, is also stable tosubsequent processes.

Preferably this test surface will have fewer optical layers, but morecomplex test surfaces possessing more layers corresponding to theformulae provided in Table 3 with modifications discussed below. Asalready noted, the theoretical calculations are the starting point formaterial selection. Theoretical considerations may be used to determinewhich materials are compatible with a pre-selected substrate. Thecoating thickness may be set at the predetermined quarterwave thicknessor to a preselected interference color. However, for the construction ofa specific binding material optical film composite of this invention anumber of adjustments are required to the initial coating. Theseadjustments are described below.

For example, a substrate such as a polished silicon wafer has arefractive index of approximately 4.1. To maximize the utility of thetest surface in accordance with the first equations in Table A, theoptical thin film material selected should have an index of refractionof 2.02 (i.e., the square root of 4.1). Maximal "apparent" color changeis achieved for silicon with materials having refractive indices near2.0, such as silicon nitride (Si₃ N₄) or silicon/silicon dioxidecomposites. Other optical thin film materials that have a similarrefractive index include, but are not limited to: tin oxide, zinc oxide,chromium oxide, barium titanate, cadmium sulfide, manganese oxide, leadsulfide, zinc sulfide, zirconium oxide, nickel oxide, aluminum oxide,boron nitride, magnesium fluoride, iron oxide, silicon oxynitride(Si_(x) O_(y) N_(z)), boron oxide, lithium fluoride, and titanium oxide.

Silicon Nitride

One method for the deposition of silicon nitride is a plasma-enhancedchemical vapor deposition technique similar to that described above forthe deposition of a-Si:H. It is recognized that this technique, ormodifications of this technique, are suitable for the deposition of alarge number of materials. For example, to produce Si₃ N₄, ammonia (NH₃)gas is added to silane gas. Silicon nitride performs well as an opticalthin film on substrates of monocrystalline silicon and polycrystallinesilicon, or on amorphous silicon and polycrystalline silicon onoptically passive substrates.

The compatibility of the silicon nitride deposition process with thea-Si:H deposition process produces a very cost-effective combination.The two films may be deposited as follows. Glass substrates are mountedin an evaporation systems where a 2000 Å thick layer of aluminum isdeposited on the glass, as described above. Then the substrates aremounted in a plasma-enhanced chemical vapor deposition system, where a 1micron thick layer of a-Si:H is deposited, as described above, followedby a silicon nitride layer. In this way an inexpensive reflection-modetest surface is formed on a glass substrate. This approach may beextended to the deposition of these coatings on dielectrics and flexiblesubstrates described in U.S. Pat. No. 3,068,510 issued Dec. 18, 1962 toColeman.

The refractive index of the silicon nitride, or by analogy thesilicon/silicon dioxide composites, may be controlled in the vapordeposition process. The ratio of gases may be varied, or the depositionrates may be varied, and a variety of other methods known to thoseskilled in the art may be used to control or select the refractive indexof the optical thin film deposited.

Multi-layer Films

Multi-layer optical thin film coatings may be deposited by electron beamevaporation. A substrate is mounted in a vacuum deposition chamber, andsuspended over two or more crucibles of the various material to beevaporated. Each crucible is then heated by an electron-beam gun, andthe rate of evaporation monitored using a crystal thickness monitor.Each crucible is covered by a movable shutter. By alternately openingand closing the shutters, the substrate is exposed sequentially to eachvapor stream, until the desired multi-layer stack has been deposited, ora multi-component film is deposited. The described procedure may begeneralized to more than two crucibles in order to deposit multiplelayers of various optical thin film materials, or multi-component filmstailored to a specific refractive index.

The test surface when coated at a specific thickness with a siliconnitride film suppresses certain wavelengths in the blue range of visiblelight and therefore reflects a yellow-gold interference color. Althougha yellow-gold interference color is utilized in the examples below, theinterference color of the test surface can be any suitable color in thespectrum of light. The color depends on the substrate material selected,the chemical composition and refractive index of the optical layer/sselected, and the thickness and number of coated layers. These designtechniques can also be utilized to produce test surfaces with signals orbackgrounds in the ultraviolet or infrared region of the spectrum oflight, however, these test surfaces are useful only in instrumenteddetection of a bound analyte.

For example, lithium fluoride may form one component of a multi-layerstack. It has a refractive index of 1.39 for visible light, and thusforms a one-quarter wavelength layer for green light at a thickness of925 Å. It may be evaporated from a platinum crucible at approximately900° C.

Titanium Film

Titanium films are particularly useful for the production of opticalfilms. Such films have advantages since they use materials which aresafer to handle and dispose of than other optical materials, such asSiH₄. The method of application is also more cost effective and rapidwith less instrumentation required.

Titanium dioxide has a refractive index of approximately 2.2 for visiblelight, and thus forms a one-quarter wavelength layer for green light ata thickness of 585 Å. Because titanium dioxide decomposes into loweroxides upon heating, the evaporated films are not stoichiometric. Todeposit stoichiometric titanium dioxide the electron-beam must bepulsed. The deposition occurs at approximately 2000° C.

Organotitanates may be hydrolyzed to titanium dioxide (TiO₂) underconditions which prevent premature polymerization or condensation oftitanates. The latter reactions are base catalyzed. The organotitanatemay be mixed with an aqueous solvent system and a surfactant. Thesolvent/surfactant system selected should tolerate a high solid content,have good leveling or spreading capacity, and be miscible with water.Alcohols and the fluorosurfactants manufactured by 3M (Minnesota) areparticularly useful for this method. Hydrolysis of the organotitanateshould occur prior to any polymerization or condensation, and thesolvent system should be acidic to prevent undesired polymerizationreactions. The counter ion supplied by the acid can be used to improvethe solubility of the titanium--acetic acid and hydrochloric acid arepreferred. A nonaqueous solvent system may be used but theorganotitanate must not be pre-hydrolyzed. The solvent must be anhydrousto improve the stability of the coating solution. Suitable solventsinclude toluene, heptane, and hexane. A surfactant is not required (asin the aqueous solvent system), but may further improve the coatingcharacteristics.

Once the organotitanate and the solvent system are mixed, apredetermined volume of this solution is applied to an optical substrateusing a spin coating technique. When the organotitanate is mixed with anon-aqueous solvent system the solution is applied to the opticalsubstrate by dynamic delivery. In a dynamic delivery method thesubstrate is attached to the spin coater and spun at 4,000 to 5,000 rpm.The solution is applied to the spinning substrate which continues tospin until an even film is obtained. For aqueous solvent systems,dynamic or static delivery of the solution is possible. In staticdelivery, the solution is applied to the substrate and then the spinningis initiated. The spin rate required is dependent on the percent solidsin the solution, the volume applied to the substrate, and the substratesize. The thickness of the titanium layer generated is a function of thepercent solid, the volume applied, and the spin rate.

The titanium dioxide layer may be cured to the substrate by a number oftechniques. The refractive index of the titanium dioxide layer iscontrolled by the temperature of the substrate during curing and to amuch lesser degree the length of the curing process. The curing processmay use a furnace, an infrared heat lamp, a hot plate, or a microwaveoven.

Titanium dioxide offers a number of advantages for this application:

1. It is inexpensive and easy to apply to a wide range of opticalsubstrates and is not hazardous to produce.

2. Its refractive index can be controlled and will cover a range from1.6 to 2.2. Thus, it can be used to give an equivalent material tosilicon nitride with a refractive index of 2.0.

3. The titanol formed at the surface reacts chemically similar tosilanols in subsequent derivatization processes (see below).

In addition to the titanates, silicates, aluminum alkyloxides, and thecorresponding analogs of zirconium may all be used to produce an opticalthin film by this method.

In addition to spin coating the titanium dioxide, polysilazanes may beused to produce silicon nitride coatings by spin coating. Theseprotocols may also be adapted for use in this technology. T-resins suchas polymethylsilsesquioxane or polyphenylsilsesquioxane (general formulaRSiO₁.5) may be spin coated to the optical substrate or support toprovide a silicon carbide surface with a suitable refractive index forgeneration of an optical thin film.

Optimization Procedure

A model was developed to select an optimal background interference colorfor any particular combination of substrate, optical thin film (ARfilm), attachment layer and receptive material. Since the mathematicalmodels developed to date are not effective to provide useful devices ofthe present invention, these models are used only as a starting point inthe device construction. Optimization is necessary to provide a deviceof this invention. For illustration purposes only, the selectedsubstrate was a silicon wafer and the optical material selected wassilicon nitride. The most highly contrasting colors observed were aye0llow-gold changing to magenta with an increase in mass on the testsurface.

Referring to FIGS. 3A-G, a method for selection of the optimalthicknesses of each layer for a device of the present invention isdisclosed for a silicon nitride film on silicon. In a first step (seeFIG. 3A), a silicon substrate is provided either with a specular ornon-specular surface. A silicon nitride film is provided on this surfaceand, as shown in FIGS. 3B and 3C, is eroded away in a stepwise fashionby heating and stirring in an appropriate solution. The timing of eachstep is selected such that the portion which is subjected to erosion forthe longest period of time exhibits a pale gold color, while thatportion which is not exposed to erosion exhibits a deep blue color (seeFIG. 3D). An attachment layer and a receptive material layer foranalytes to be detected are provided on the silicon nitride (see FIGS.6E and 6F). These layers are provided in a thickness which may bedetermined empirically, or can be similarly optimized (e.g., in thisstepwise fashion) if so desired. In a seventh step, an assay isperformed with three portions of the strip being treated in a differentmanner such that a negative response, a weak response, and a strongresponse can be recorded. The results are shown in FIG. 3G, and thethickness of silicon nitride useful in the invention can be determinedby those sections providing the strongest weak positive response in thetest.

Specifically, a silicon wafer was prepared with a thick coating (800 Å)of silicon nitride so that the wafer appeared to be a deep blue. Thenthe optical thin film material was etched off the wafer in a hot,phosphoric acid bath to produce a wedge of interference colors. Theoptical material was etched such that 300 Å remained at one end of thewedge and 700 Å remained at the other end of the wedge. (At 180° C. thesilicon nitride was removed at approximately 20 Å per minute.)

The etched, wedged test surface was coated with an attachment material,and then a receptive material. The reactive surface was analyzed with anegative, a weak positive, and a strong positive sample. The thicknessof the optical material was then measured at the wedge segments whichappeared to provide the most distinctive color change, or visualcontrast. The optimal film thickness is most readily selected based onthe composite test surface analysis. This process maximizes the visualcontrast obtained for the specific assays.

Silicon nitride is easily etched to produce the wedge of thicknessesneeded for this empirical evaluation. Many materials are susceptible toan acid etching or base etching process. Other chemical methods ofetching the material are possible. If a desired optical film is noteasily removed from a particular optical substrate because the film istoo easily destroyed, or the optical substrate is not stable to therequired etchant, another method of generating the wedge may be used.For instance, monocrystalline silicon is not stable to prolongedexposure to basic solutions. If an optical film on silicon requires abasic etchant the wedge can not be generated using a chemical approach.

Several alternatives exist: (1) the optical film may be deposited on anoptical substrate which is introduced stepwise into the coating chamberover a period of time. Each newly exposed section will receive a thinnercoating than the previously exposed section. (2) The substrate may bemasked and the mask removed stepwise over a period of time. (3) Severaldifferent coating runs each producing a different thickness of opticalmaterial may be performed. (4) Ion milling may also be used to etchcertain materials.

For any given optical substrate and a substitute optical thin film ofthe same refractive index as the original optical thin film, thisoptimization need not be repeated. The above method was used toestablish that a 480-520 Å film of Si₃ N₄, with a refractive index of2.0, was required for a silicon wafer (optical substrate) to be used ina binding assay (see Example 2). It has been demonstrated that TiO₂ at arefractive index of 2.0, using the same attachment layer and receptivematerial, requires a 480-520 Å coating. Minor thickness adjustments maybe required if the refractive index is not exactly that of the originalmaterial.

Thus, the formulae established for the coating of optical thin films areused as a guideline only for the production of a test surface suited toa specific binding assay. For a pre-selected substrate, the square rootdependence of an optical thin film is used to screen appropriate opticalmaterials. Some deviation from the perfect square root dependence isacceptable for this invention. The use of a quarterwave thickness of theoptical coating is only an initial guide to coating thickness. Thicknessof the optical thin film must thus be empirically derived inconsideration of the specific binding materials. The composite specificbinding optical thin film of this invention does not meet the conditionstheoretically required to produce such a film. Neither the thickness northe refractive index rules are followed. Surprisingly such deviationfrom these accepted formulae results in a test surface which is verysensitive to mass changes or thickness changes.

While of less importance, the relative thicknesses of each layer, andnot just the optical thin film layer, may be varied as described aboveto optimize the final test device for any particular attachment layerand receptive material layer.

Attachment Layer

This invention is further concerned with materials and methods forproducing a layer which attaches the specific binding layer to theoptical substrate or optical thin film. Specifically, the inventionpertains to a method for producing an attachment layer which optimizesthe functional density, stability, and viability of receptive materialimmobilized on that layer. The attachment materials selected must becompatible with the biological or receptive materials, must physicallyadhere or covalently attach to the upper test surface (whether anoptical thin film is included or not), must preferably not interferewith the desired thin film properties of the test surface, and must besufficiently durable to withstand subsequent processing steps.

The density and stability of immobilized receptive material (or, in somecases, enzymes) must be controlled to optimize the performance of anassay test surface.

Applicant has determined that one problem in obtaining useful devices ofthis invention was the extremely limited macroscopic and/or microscopicsurface area of the test films employed in a thin film assay as comparedwith the microscopically convoluted surface characteristics of otherconventional solid phase assay materials. In most cases, the opticalsubstrate must be evenly coated with a continuous attachment layer thatprotects the receptive material from any toxic effects of the reflectivesubstrate while adhering it to the surface.

In conventional solid phase assays, the larger test surfaces generallyemployed, such as microtiter wells, have much greater total surface areaand microscopically convoluted surfaces relative to a thin filmsubstrate. Thus, the amount of receptive material immobilizedcompensates for any sparsity in coverage, or any losses in viability(ability to bind analyte) which result from conformational or chemicalchanges caused by the immobilization process. It also compensates forany receptive material which may be unavailable for binding due to poororientation. Thus, applicant has discovered that in direct thin filmassays the surface area limitations require the use or development ofspecial materials and procedures designed to maximize the functionaldensity, viability, stability, and accessibility of the receptivematerial.

Much of the original work to adapt siliceous materials for retention ofspecific binding molecules originated with affinity chromatographyapplications and used silica (SiO₂) gel, and solid supports such asglass. Initial activation of silica towards the binding material wasaccomplished by treatment with a dichlorodimethylsilane. Silanization,regardless of the process used to apply the silane, can introduce groupscapable of covalently attaching the molecule by chemical means.

Previously, optically active surfaces have been made hydrophobic by useof dichlorodimethylsilane (C₂ H₆ Cl₂) which bonds with hydroxyl groupson the surface of silica to attach two methyl groups to that surface.Thus, a slight hydrophobicity results. Applicant has determined thatsuch a reaction does not produce an optimal surface reactivity.Referring to FIG. 4, there is shown in diagrammatic form the bonding ofa more useful silane material which bonds to the silica groups presenton a substrate. Only bonding of such silane molecules to the surfaceprovides an available group, such as an amine group for bonding with areceptor molecule. It is evident from this figure that the greatestnumber of biological molecules that can be bonded in this way is equalto the number of silica groups available for interaction with silane. Incontrast, as shown in FIG. 5, even more useful attachment molecules ofthe present invention are multivalent with respect not only to thesilica groups present on a substrate (thus, providing a stronger bond),but are also multivalent with respect to the groups that can bond to thereceptor molecules since each R group shown in the figure can bemultivalent, and can even bond with further siloxanes, if so desired.Those of ordinary skill in the art can readily determine equivalentsiloxanes or other molecules which can be used as attachment layers toincrease the amount of receptor molecules that may be bonded on anyparticular silicon-containing or other substrate.

Applicant has discovered that when silicon replaces silica as the solidsupport or substrate for subsequent attachment, conventionalsilanization is inadequate in this invention. A silane requires thepresence of silanol residues in order to attach to the surface. Withmonocrystalline silicon, the silanol density is insufficient to yieldthe density of functional groups desired for immobilization reactions,thus, less than optimal receptive material will be attached to the testsurface, see FIG. 4. Silica and many glasses possess a high silanolcontent or are easily treated to provide a high silanol content.However, silicon also introduces surface effects, not observed withsilica or glass, which are toxic or detrimental to biomolecules. Suchsilanization processes also produce hazardous materials which requiredisposal, and in many cases are tedious and difficult to monitor andcontrol. While this silanization process provides some level ofreactivity, it does not provide the level of sensitivity required formany applications. In addition, amine-containing silanes introduce anumber of unique difficulties. One is that amine-functionalized silanesare water soluble and the amine group catalyzes the hydrolysis of thesilane from a modified surface. Applicant has discovered that silanesmodified with a polymer are functionally better in devices of thisinvention, e.g., PEI modified silane or its equivalent (FIG. 5).

In a preferred embodiment, the attachment layer is spin coated oraerosol spray coated in a uniform manner. The various intermediatematerials are coated to the substrate at thicknesses between 5 Å and 500Å (thicker amounts can be employed). The layer can be formed of anymaterial that performs the following functions and has the followingcharacteristics: creates a favorable environment for the receptivematerial, permits the receptive material to be bound in active,functional levels (preferably by a cost-effective method), adherestightly to the optical substrate, and can be coated uniformly.

Ideally, for direct eye detection methodologies, the surface activationtechnique should provide a covalent modification of the surface forstability while introducing a very dense uniform or conformal film onthe surface of the substrate. A strongly adsorbed conformal film withoutcovalent attachment may be adequate, for example, suitable substrates,such as monocrystalline silicon, macroscopically planar, uniform opticalglasses, metalized glass and plastic, whether or not coated with anoptical layer (i.e., SiO, SiO₂, Si_(x) N_(y), etc.) have a deficiency ofavailable reactive groups for covalent attachment, but are useful inthis invention. Once applied, the attachment layer should provide anenvironment which supports the adherence of a specific binding layer bycovalent or adsorptive interactions, that is dense and functional. Thisattachment layer must be of sufficient thickness to separate thespecific binding layer from any toxic effects of the initial opticalsubstrate.

Three examples of types of materials that may be used for the productionof this test surface are now described. Referring to FIG. 5, non-linearbranched polymeric siloxanes may meet the requirements of covalentattachment to the substrate and will adhere receptive material in areactive and stable film. These polymers typically contain 2-3 branchpoints which may introduce a number of different functionalities (R) tothe surface; including aminoalkyl, carboxypropyl, chloropropyl,epoxycyclohexylethyl, mercaptopropyl, phenethyl, phenethylsulfonate,vinyl, methyl and methacryloxy-propyl (produced by Petrarch Systems).These polymeric siloxanes are particularly useful when a layer ofsilicon nitride is the upper surface as even fewer silanols areavailable for attachment. T-structured polydimethylsiloxanes withfunctionality at the branch terminus include carboxy, propyl, and vinylgroups (Petrarch Systems). Typical examples of these materials areprovided in U.S. Pat. Nos. 4,208,506, 3,530,159 and 4,369,268, allhereby incorporated by reference herein.

A second group of materials which demonstrate utility in the productionof these test surfaces are copolymeric, conformal, surface activator orfilm forming latexes which commonly consist of a styrene/polybutadienemixtures. Although these preparations perform as particles while insolution, they do not retain their particulate nature at a surface orupon drying. These materials are designed to strongly adhere to surfacemicro-structures. TC7 and TC3 particles distributed by Seradyn and theSurface Activators (amide or carboxylic acid) distributed by BangsLaboratories or Rhone-Poulenc are particularly well suited to thisapplication. Any similar film forming latex or styrene/butadiene/othercopolymer may be used.

Another class of compounds which has utility in the production of thistype of test surface is dendrimers, or star polymers, or molecularself-assembling polymers. These polymers strongly adhere to a surfaceonce dried. These materials are generated in a cyclic fashion, eachcycle producing a new generation of material. Any generation of materialmay be used in this application, however, generation 5 (showndiagrammatically in FIG. 18) has been demonstrated to provide the bestreactivity. These materials are produced and composed of the materialslisted in U.S. Pat. Nos. 4,507,466; 4,588,120; 4,568,737; and 4,587,329.Representative of a ternary dendrimer is the polyamidoamine shown inFIG. 18. In this figure, Y represents a divalent amide moiety. Such as--CH₂ CH₂ CONHCH₂ CH₂ -- and YN is a repeating unit. The terminal groupsmay be amines as shown in FIG. 17 but may be any active group which willserve as a dendritic branch for subsequent generations. Other suitabledivalent moieties include alkylene, alkylene oxide, alkyleneamine andthe like with the terminal being an amine, carboxy, aziridinyl,oxazolinyl, haloalkyl, oxirane, hydroxy, carboxylic esters, orisocyanato group.

All of these materials are stable to organic solvents which improve thespreading capacity on a solid support. This allows the attachment layerto be generated by a spin coating technique which is easy to control andproduce in volume. Alternate methods of application include dip coating,spray coating, or other aerosoling techniques. The use of an organicsolvent is acceptable because, following the curing process (generally aheat treatment), none of the organic solvent remains to contact thereceptive material. The curing process improves the adhesion of theattachment layer to the optical test surface and helps drive thepolymerization and condensation processes. Evaluation of each of thesematerials and the precise methods of producing these attachment layersare presented in Examples 5, 6, and 7, below.

The siloxanes point to a general class of materials that are useful inthis application. The siloxanes are not water soluble, and do nothydrolyze upon contact with an aqueous coating solution or sample.Because of the branched structure and the polymeric features, ratherthan forming a single isolated island as a silane will (FIG. 4), thepolymeric structure forms a continuous film (FIG. 5) on the testsurface. The highly cross-linked structure of a siloxane increases thetotal surface coverage. The refractive index of the siloxane film can becontrolled or varied with the functional group incorporated into theside chains of the siloxane and thus caused to interact with the otherlayers to produce an appropriate interference film.

The siloxanes covalently modify the substrate, but this is not anessential feature, as other materials which adhere to the surfacewithout any subsequent delamination and are stable to mechanicalmanipulation are useful. Methods of coating polymers to substrates areknown to those skilled in the art of semiconductor fabrication.

Although not required, additional materials which convey a desiredproperty may be affixed to the attachment layer. This layer couldimprove receptor material orientation, for example, use of Protein A orProtein G for orienting antibodies. Other materials which can be usedinclude avidin-biotin, synthetic or recombinant Protein A/Protein Gfragments or peptides or combined A/G peptides, etc.

The immobilization chemistry for attaching the receptive material to theattachment layer is selected based on the properties of both theattachment layer and the receptive material. The receptive material canbe covalently or passively attached to this material. When theattachment layer is specifically adapted for covalent attachment, anadditional step to activate the attachment layer may be required. Avariety of activation and linking procedures can be employed, forexample, photo-activated biotin can be employed to adhere the receptivematerial. Usually, it is sufficient to passively adsorb the receptivematerial to the attachment layer, thus avoiding the time and expense ofimmobilization chemistry procedures.

Receptive Material

The receptive material is defined as one part of a specific binding pairand includes, but is not limited to: antigen/antibody, enzyme/substrate,oligonucleotide/DNA, chelator/metal, enzyme/inhibitor,bacteria/receptor, virus/receptor, hormone/receptor, DNA/RNA, orRNA/RNA, oligonucleotide/RNA, and binding of these species to any otherspecies, as well as the interaction of these species with inorganicspecies.

The receptive material that is bound to the attachment layer ischaracterized by an ability to specifically bind the analyte or analytesof interest. The variety of materials that can be used as receptivematerial are limited only by the types of material which will combineselectively (with respect to any chosen sample) with a secondarypartner. Subclasses of materials which can be included in the overallclass of receptive materials includes toxins, antibodies, antigens,hormone receptors, parasites, cells, haptens, metabolites, allergens,nucleic acids, nuclear materials, autoantibodies, blood proteins,cellular debris, enzymes, tissue proteins, enzyme substrates,co-enzymes, neuron transmitters, viruses, viral particles,microorganisms, proteins, polysaccharides, chelators, drugs, and anyother member of a specific binding pair. This list only incorporatessome of the many different materials that can be coated onto theattachment layer to produce a thin film assay system. Whatever theselected analyte of interest is, the receptive material is designed tobind specifically with the analyte of interest. The matrix containingthe analyte of interest may be a fluid, a solid, a gas, or a bodilyfluid such as mucous, saliva, urine, fecal material, tissue, marrow,cerebral spinal fluid, serum, plasma, whole blood, sputum, bufferedsolutions, extracted solutions, semen, vaginal secretions, pericardial,gastric, peritoneal, pleural, or other washes and the like. The analyteof interest may be an antigen, an antibody, an enzyme, a DNA fragment,an intact gene, a RNA fragment, a small molecule, a metal, a toxin, anenvironmental agent, a nucleic acid, a cytoplasmic component, pili orflagella component, protein, polysaccharide, drug, or any othermaterial, such as those listed in Table A. For example, receptivematerial for the bacteria listed in Table A may specifically bind asurface membrane component--protein or lipid, a polysaccharide, anucleic acid, or an enzyme. The analyte which is specific to thebacteria may be a polysaccharide, an enzyme, a nucleic acid, a membranecomponent, or an antibody produced by the host in response to thebacteria. The presence of the analyte may indicate an infectious disease(bacterial or viral), cancer or other metabolic disorder or condition.The presence of the analyte may be an indication of food poisoning orother toxic exposure. The analyte may indicate drug abuse or may monitorlevels of therapeutic agents.

One of the most commonly encountered assay protocols for which thistechnology, can be utilized is an immunoassay. The discussion presentedfor construction of a receptive material layer below specificallyaddresses immunoassays. However, the general considerations apply tonucleic acid probes, enzyme/substrate, and other ligand/receptor assayformats. For immunoassays, an antibody may serve as the receptivematerial or it may be the analyte of interest. The receptive material,for example an antibody, must form a stable, dense, reactive layer onthe attachment layer of the test device. If an antigen is to be detectedand an antibody is the receptive material, the antibody must be specificto the antigen of interest; and the antibody (receptive material) mustbind the antigen (analyte) with sufficient avidity that the antigen isretained at the test surface. In some cases, the analyte may not simplybind the receptive material, but may cause a detectable modification ofthe receptive material to occur. This interaction could cause anincrease in mass at the test surface or a decrease in the amount ofreceptive material on the test surface. An example of the latter is theinteraction of a degradative enzyme or material with a specific,immobilized substrate, see Example 13. The specific mechanism throughwhich binding, hybridization, or interaction of the analyte with thereceptive material occurs is not important to this invention, but mayimpact the reaction conditions used in the final assay protocol.

In general, the receptive material may be passively adhered to theattachment layer. If required the free functional groups introduced ontothe test surface by the attachment layer may be used for covalentattachment of receptive material to the test surface. Chemistriesavailable for attachment of receptive materials are well known to thoseskilled in the art.

A wide range of techniques can be used to adhere the receptive materialto the attachment layer. Test surfaces may be coated with receptivematerial by: total immersion in a solution for a pre-determined periodof time; application of solution in discrete arrays or patterns;spraying, ink jet, or other imprinting methods; or by spin coating froman appropriate solvent system. The technique selected should minimizethe amount of receptive material required for coating a large number oftest surfaces and maintain the stability/functionality of receptivematerial during application. The technique must also apply or adhere thereceptive material to the attachment layer in a very uniform andreproducible fashion.

Composition of the coating solution will depend on the method ofapplication and type of receptive material to be utilized. If a spincoating technique is used a surfactant may improve the uniformity of thereceptive material across the optical substrate or support. In general,the coating solution will be a buffered aqueous solution at a pH,composition, and ionic strength that promotes passive adhesion of thereceptive material to the attachment layer. The exact conditionsselected will depend on the type of receptive material used for theassay under development. Once coating conditions are established for aparticular type of receptive material, e.g., polyclonal antibodies,these conditions are suitable for all assays based on such receptivematerial. However, chemically distinct receptive materials, for examplepolyclonal antibodies and nucleic acids, may not coat equally well tothe attachment layer under similar buffer and application conditions.

It has been demonstrated that when the receptive material is an antibodysuitable adhesion is obtained when the attachment layer is aT-structured siloxane. The T-structured siloxanes provide a very uniformhydrophobic surface for antibody interaction, see Example 6.

Surprisingly, the film forming latexes generally provide a betterattachment for antigens than do the siloxanes. Antibody interaction withthe immobilized antigen is improved on a siloxane modified surface,while enzymatic reaction with a substrate is improved on a latexmodified surface relative to a siloxane modified surface.

The materials and methods described above allow the construction of aspecific binding test surface. The test surface is composed of anoptical substrate or support, an optional optical thin film, anattachment layer, and finally a layer of receptive material. For avisual determination of a specific binding event or interaction, thecomposite interference film is actually designed to include the opticalthin film, the attachment layer and the receptive material. The initialinterference color selected must be maintained when the attachment layerand receptive material are coated onto the optical thin film, see FIGS.3E and 3F. Once a surface is coated with receptive material a small spotof a preparation containing the analyte of interest may be applied tothe surface. This is incubated for a few minutes, rinsed, and then driedunder a stream of nitrogen. This will generate a procedural controlwhich will be developed whether the sample being assayed is positive ornegative. This control assures the end-user, that the assay protocol wasfollowed correctly and that all the reagents in the kit are performingcorrectly. The procedural control may be applied in any pattern desired.

Like the procedural control the receptive material may be applied in apattern. Thus, the device will provide a symbol detectable by eye inresponse to polychromatic light when the optical thin film is applied tothe optical substrate. The coating solution containing receptivematerial may be applied to the surface which is covered with a mask. Themask will allow the receptive material to be immobilized on theattachment layer only in the sections which are exposed to the coatingsolution. A surface which is uniformly coated with receptive materialmay be covered with a mask and the receptive material may be selectivelyinactivated. There are a number of techniques which are suitable for theinactivation of receptive material. One of the simplest techniques forbiological materials is to expose section of the receptive material toUV irradiation for a sufficient period of time to inactive the material.The mask may be designed in any pattern which will assist the end-userin interpretation of the results.

Techniques such as stamping, ink jet printing, ultra-sonic dispensers,and other liquid dispensing equipment are suitable for generation of apattern of the receptive material. The receptive material may be appliedin the pattern by these techniques, incubated for a period of time, andthen rinsed from the surface. Exposed sections of attachment materialmay be coated with an inert material similar to the receptive material.

A particularly useful combination of interference colors relies on ayellow/gold interference color for the test surface background orstarting point. Once an increase in mass occurs at the surface, massbeing a direct function of thickness and concentration, the reacted zonechanges interference color to a purple/blue color. As described above,the optical thin film can be adjusted and optimized to compensate forthe layers required in the construction of the biological test surfaceto maintain the desired starting interference color.

Mass Enhancement

Thin film detection methods which provide direct determination ofspecific binding pairs offer significant advantages relative toradioactive or enzymatic means, including fluorescent, luminescent,calorimetric, or other tag-dependent detection schemes. Thin filmsystems can be applied in the detection of small molecules. Suchanalytes, however, fail to produce sufficient thickness or opticaldensity for direct eye or instrumented detection. Applicant hasdiscovered that a means for mass enhancement is necessary. Thin filmdetection systems, however, perform optimally when the integrity of thefilm is maintained. Thus, any method designed for amplification in sucha system should provide an increase in thickness or mass and maintainthe film integrity, as well as meet any limitations imposed by thedetection systems, and should be of the simplest possible construction.

C. Fredrik Mandenires and K. Mosbach, 170 Anal. Biochem. 68, 1988,describe a method of using small silica particles coated withconcanavalin A or an anti-IgG antibody in an ellipsometric assay. Silicaparticles provide a refractive index which is sufficiently close to thebiological layers that they increase the apparent thickness of thebiological layers in a concentration dependent manner. These particlesbeing rigid in nature do not, however, maintain the integrity of thefilms. Thus, light scattering occurs.

The amplification technique may be directly related to the concentrationof the analyte of interest or may be inversely proportional to theconcentration of the analyte of interest as in a competitive orinhibition assay format. The binding of a mass enhancement oramplification reagent must be a specific function of the analyte bindingto the test surface and may be considered as part of a signal generatingreagent.

Referring to FIGS. 7A-E, there is shown in diagrammatic form two methodsby which the presence of an analyte on a device of the present inventioncan be detected by signal amplification. For example, the signal may beamplified by contacting the receptive material with analyte labeled witha latex particle or other means which will enhance the thickness of thereceptor analyte layers when the two are bound together. Alternatively,the analyte may be labeled with an enzyme, through a secondary bindingagent, such that, while the receptor-analyte-enzyme combination may notbe detectable, by provision of a substrate for that enzyme, a product isdeposited on the test device and can be detected by eye. Applicant hasfound that it is advantageous to ensure that the surface of the deviceis charged so that deposition of the product from the substrate isaided.

The mass enhancement reagent must be capable of passive or covalentattachment to a secondary receptive material. An example of passiveattachment to a mass enhancing reagent is the adsorption of antibodiesonto surface activator particles. An example of the covalent attachmentof a mass enhancing reagent to the secondary receptive material is theconjugation of horseradish peroxidase (HRP, or another enzyme) to anantibody. Regardless of the mechanism employed, the mass enhancementreagent should form a stable product or adduct with the secondaryreceptive material. The coupling protocol selected should not leave orintroduce non-specific binding effects at the test surface. The massenhancement reagent may also be capable of direct, specific interactionwith the analyte.

Thus, the invention features methods for the amplification of signals inassay systems which rely on a thin film detection method. Such methodsinclude, but are not limited to, ellipsometry, interference effects,profilometry, scanning tunneling microscopy, atomic force microscopy,interferometry, light scattering, total internal reflection, orreflectometric techniques. The materials selected for use in these typesof systems preferably maintain some degree of particulate character insolution, and upon contact with a surface or support form a stable thinfilm. The film is preferably conformal to the test surface to maintainthe desired smoothness or texture of the substrate. The characteristictexture of the surface will be dependent on the detection methodemployed. The material selected must also be capable of adhering,through covalent or passive interaction, a receptive material or onemember of a specific binding pair. A secondary receptive material orbinding reagent preferably is adhered to the signal amplifying materialor particle in a manner which preserves the reactivity and stability ofthat secondary receptive material. The secondary receptive materialapplied to the particle may be identical to, or matched to the receptivematerial immobilized on the test surface. The combination of a secondaryreceptive material or binding reagent and additional material, whether aparticle, an enzyme, or etc., forms a mass enhancement or signalgenerating reagent.

In general, an optical assay that requires amplification consists of asubstrate whose properties and characteristics are determined by thetype of detection method used, an optional secondary optical material,an attachment layer, a layer of receptive material, and the massenhancement reagent. A general assay protocol requires that the samplesuspected of containing the analyte of interest be processed through anytreatment necessary, such as extraction of a cellular antigen, and thenbe mixed with the secondary or amplification reagent. An aliquot of thismixture is applied to the receptive material coated substrate. After anappropriate incubation period, the unbound material is separated fromthe reacted film by either a physical rinse/dry protocol or with adevice contained rinse/dry step. The signal is then interpreted by eyeor instrumentally. The introduction of the secondary or amplificationreagent can be achieved by addition of a reagent to the sample, as alyophilized material in the sample collection or application device, orembedded in an assay device.

Polymer Solid

Polymers useful in this invention are conformal (film forming) and donot introduce a particulate character to the surface. A wide variety ofstyrene-butadiene copolymers have found utility in agglutination assays,immunoassays, and chromatography applications. The latexes commonly usedare highly cross-linked, rigid copolymers. The most common use of latexparticles in these applications is as the solid support for capture andseparation of the desired analyte. Styrene-butadiene copolymers with lowcross-linking have been designated surface activators or film forminglatex particles. These preparations behave as particles in solution butupon contact with a surface dry to a conformal film. These film formingstyrene-butadiene copolymers may contain a wide variety of functionalbinding groups.

Rigid polystyrene particles also have been used for signal generation byincorporation of a dye. These particles are simply an alternate methodfor the introduction of a tag or label for signal generation. Use ofcolored or dyed latexes for agglutination assays and for membrane basedassays have been extensively utilized (for a review see L. B. Bangs,American Clinical Laboratory News, May 1990). As in previous cases, theprimary requirement for these particles is that they maintain theirstructure for visualization in agglutination assays and do not distortto block the pores of the membrane-based tests. While covalentattachment provides specific advantages with certain interactingspecies, passive adsorption of the receptive material to the latex isfrequently adequate.

The production of suitable amplifying film-forming latex particlesrequires the selection of a film-forming particle or surface activatorcompatible with the secondary reactive species and of sufficient size toincrease the apparent thickness or density of the captured analyte.

The secondary reactive species may be immobilized on the surfaceactivator particles by incubation at the appropriate temperature for aperiod of time. The temperature selected will be influenced by thechemistry utilized to attach the secondary reactive species to theparticle, the nature of the reactive species, and the composition of theparticle. In addition to the temperature, length of incubation, andchemistry of immobilization, the buffer composition (pH and ionicstrength), and the amount of secondary reactive material must also beoptimized to the particular application.

The specific examples (14 and 15) given below are intended to beillustrative of the type of method(s) used for the production of thefilm-forming amplification reagent. The conditions described are notintended as a limitation in the preparation of such amplificationreagents. The styrene/butadiene/vinyl copolymers are the preferred filmforming latex compositions. However, any styrene/butadiene copolymerwhich maintains the film forming property is acceptable. The functionalgroup may be of any chemical composition which will support the adhesionor interaction of a secondary binding reagent, where the secondarybinding reagent will specifically bind with the analyte of interest. TheTC7 and TC3 formulation distributed by Seradyn and the Surface Activatorformulations distributed by Bangs Laboratories or Rhone-Poulenc arepreferred (the catalogs of which are hereby incorporated by referenceherein). More conventional latex particles (referred to as S/B/V-CONH₂,S/B/V-COOH, S/V-CONH², S/R-NH₂, S/HYDRAZIDE, S/V-COOH, S/B-COOH,S/B-CONH2, PS, S/VBC, S/A/V-COOH, PMMA-COOH, S/A-OH, S/R-OH, andS/R-SHO) have demonstrated some utility in this invention, but tend toproduce a more diffuse signal than the film forming latexes.

Catalytic Production of Solid

Applicant has found that even more sensitive optical thin film assayscan be obtained with an enzyme/substrate pair which produces insolubleprecipitated products on the thin film surface. The catalytic nature ofthis amplification technique improves the sensitivity of the method.Enzymes which are useful in the present invention include glucoseoxidase, galactosidase peroxidase, alkaline phosphatase and the like.However, any process which provides a specific component which can beattached to a receptive material and can catalyze conversion of asubstrate to a precipitated film product is suitable to this technology.An insoluble reaction product results when immobilizedantibody-antigen-antibody-HRP complex is present on the test surface.The product is precipitated by the action of a precipitating agent suchas combination of alginic acid, dextran sulfate, methyl vinylether/maleic anhydride copolymer, or carrageenan and the like, and withthe product formed by the interaction of TMB(3,3',5,5'-tetra-methyl-benzidine) with an oxygen free radical. Thisparticular substrate will form an insoluble product whenever a freeradical contacts the TMB. Other substances such as chloronaphthol,diaminobenzidene tetrahydrochloride, aminoethyl-carbazole,orthophenylenediamine and the like can also be used. These are used inconcentrations from about 10 to about 100 mM. It is by these means thata measurable increase in mass occurs with the enzyme-conjugate layer.The color signal is unaffected by the underlying color of anychromophore present in the substrate solution. A variety of enzymesubstrate systems or catalytic systems may be employed that willincrease the mass deposited on the surface.

Examples of such an enzyme-labeled antibody methods in thin film assaysfor the detection of low levels of the polysaccharide antigens derivedfrom the group of bacteria commonly responsible for bacterial infectionsin man, such as Meningitidis and Streptococcus are presented in Examples16, 17, 18.

Referring to FIGS. 6A-F, there is a graphic representation of across-section of the multilayer device having a substrate upon whoseupper surface, various layers are coated. In one example, these layersinclude a layer of silicon nitride immediately adjacent to the upperoptical substrate layer, an attachment layer such as a polymericsiloxane, and the receptive material, which for a bacterial antigenassay is an antibody. Referring to FIGS. 7C-E, when the analyte ispresent, a complex with the enzyme-labeled antibody and analyte issimultaneously formed on the test surface. It is over this mass that thesubstrate is added to cause the product precipitate described to form.

If desired, the analyte of interest may be combined with the massenhancing reagent and the immobilized receptive material either in asimultaneous or sequential addition process. Either mechanism results inthe formation of an analyte/mass enhancement reagent complex which isimmobilized on the test surface. Thus, the mass enhancement reagent maybe mixed directly with the sample. This mixture may then be applied tothe reactive test surface and incubated for the required period. This isa simultaneous assay format.

In some cases additional sensitivity is gained by performing asequential addition of the sample followed by the mass enhancementreagent. Any mechanism or specific interaction can be exploited for thegeneration of a mass enhancement reagent. For instance, nucleic acidsare known to tightly bind or intercalate a number of materials, such asmetals, and certain dyes. These materials would serve to introduce massinto a specifically immobilized nucleic acid.

The increase of the product layer may be determined by various meansincluding a visual means or by the use of instrumentation, such asellipsometry and where light intensity differentials are caused by theincreased thickness. The receptive material enzyme complex is thuscapable of direct interaction with the analyte of interest and moreparticularly is evidence of an analyte, such as an antigen. This changeis detectable by measuring the optical thickness and does notnecessarily depend on any light reflectivity of the substrate material.One such instrument is the Sagax Ellipsometer, described in U.S. Pat.Nos. 4,332,476, 4,655,595, 4,647,207, and 4,558,012, which disclosuresare incorporated in full and made a part hereof.

Devices

Several configurations of the above multilayer test surface in a deviceformat are possible. The simplest assay format is a single use, singlesample device. A more complicated device allows for a single sample tobe screened for the presence of multiple analytes. Additional devicesallow multiple samples to be screened for a single analyte or batchtesting.

The single use device provides an easy to use format which is adaptableto a wide range of assays, such as infectious disease testing, pregnancyor fertility testing, etc. Protocols for using these single test devicesare very simple. The sealed device is opened exposing the reactive testsurface. Sample is applied to the test surface and incubated for a shortperiod of time, for example, 2 minutes. The sample may or may notrequire pre-treatment, such as antigen extraction from bacteria, etc.Addition of a secondary reagent to the sample prior to application tothe test surface may also be required. Once the incubation period iscomplete, the unreacted sample is removed with a water rinse. The deviceis blotted to dry the test surface. Depending on the test and the massenhancement/amplification method used, the assay is complete or theassay may require additional incubation/wash/dry cycles. The test deviceand protocol are well suited to physician office, clinical laboratory,home or field testing environments. A protective shell is preferablyprovided around the device, e.g., composed of polystyrene,polypropylene, polyethylene, or the like, which is readily formed into amolded or injection molded devices. Multi-analyte or multi-sampledevices may be made of similar materials using similar processes.

Single Use Device

Specific examples of such devices are shown in the Figures.

For example, referring generally to FIGS. 8A-8G, a single use device maybe packaged in any size of molded device, but in this example has alength of 1.74 inches and a width of 2.22 inches and a depth of 0.375inches for the closed device. The device is constructed of a base whichwill hold the test surface and an absorbent pad for retaining the washsolution and excess sample. The device lid is designed to hold a blotterpad which will remove excess moisture from the test surface. To securethe absorbent pad in the base of the device or the blotter in the lid athin sheet of plastic is attached to each portion of the device by aliving hinge. The base and the lid of the device are joined by a livinghinge. However, any clasp or hinge combination which will allow multipleopening/closing cycles is acceptable. Once the absorbent pad and blotterare placed in the device these covers are closed to secure thematerials. These plastic covers provide protection to the end-user bypreventing exposure to the wash solution and excess sample containedwithin the absorbent materials. The lid of the device may be designedwith or without a clasp but it is preferred that a tight seal beobtained. The device should be easily disposed of or of a convenientsize for storage. All components of the device, except the reactive testsurface, may be sterilized if required.

The upper blotter pad has several unique requirements. The compositeblotter material must be set in the lid of the device directly over thetest surface. When the lid is closed and the rinsed test surface iscontacted by the blotter pad, the test surface must compress into theblotter pad sufficiently to preferentially blot solution vertically awayfrom the test surface. The blotter pad may be mounted on a small plasticslide such that fresh, dry material is presented to the test surface foradditional wash/dry steps. The initial material in the blotter isselected to rapidly wick water vertically from the surface, i.e., thepaper has a high rate of absorption, while the subsequent materials havehigh absorptive capacity and will remove solution horizontally from thetest surface. The blotter material next to the surface must not shed orscratch the optical test surface. Whatman's Grade 1Chr paper serves thisfunction very well but alternate materials are acceptable. Any highlyabsorbent material may be used as the additional layers in the blotterpad. Two additional pads in combination with the Grade 1Chr layer havebeen found to be optimal for a two stage drying process. The layers maybe free or laminated together. When a multi-step rinse/dry is required,the slide supporting the blotting material has a handle for positioningfresh blotter over the test surface. This handle fits into openings inthe protective shield over the base of the device to prevent the blotterfrom moving when it is contacting the test surface.

The reactive test surface is mounted on a pyramidal shaped pedestalwhich extends above the base of the device. Rinse solution flows overthe test surface and down the faces of the pedestal where it is trappedin the adsorbent pad. The pedestal also positions the test surface sothat it is compressed into the blotting material when the lid is closed.The test surface mounted on to the pedestal may range in size from 0.5cm² to 1.0 cm² with 0.75 cm² being preferred. The only limitation on thesize of the test surface for an eye-visible assay is that some unreactedtest surface be visible for contrasting to the reacted zone. As theinterference color change or other signal produced for a positiveresponse is permanent, the test device may be sealed and stored as apermanent record.

Referring to FIGS. 8A-8G, a single use device of the present inventionis shown. Specifically, device 20 is formed of a readily moldable hardplastic material (with a clip 22) to prevent damage to the test surfacepresent within the device, and to ensure appropriate alignment ofcomponents of the device. The lower surface of the device is indented atindent 24 such that the test surface is raised relative to otherinternal components (as shown in FIG. 8F), in which a test surface 26 israised on a pyramidal structure 28. A hinge 30 is provided on the edgeopposite clip 22 to allow raising and lowering of the upper half 32 ofthe device relative to the lower half 34.

Referring specifically to FIG. 8E, test surface 26 is provided in lowerhalf 34 of the device raised on a pyramid 28 as discussed above, suchthat liquid placed on surface 26 may flow from that surface and downpyramid 28 into an enclosed area beneath plate 36 which has an uppersurface located at the same height as the containing wall 38. Plate 36is attached by a hinge 40 to one side 42 of lower half 34. Within plate36 are provided two apertures 43 and 44.

Upper half 32 of the device is provided with a second plate 46 which isalso attached by a hinge 48 to upper half 32 along one edge 50. Twoapertures 47 and 49 are provided within plate 46. Beneath plate 46 isfilter paper 52 along with a movable plate having a handle 56 which canbe moved, as shown by arrow 58, from a position on the right hand sideof aperture 48(I) to a left hand position within that aperture (II).Such movement causes movement of filter paper 52.

Referring now specifically to FIG. 8G, plates 36 and 46 can be removedfrom their position shown in FIG. 8E by rotation about hinges 40 and 48,respectively. Beneath plate 36 is a thick filter pad (absorbent) 60designed to absorb liquid passing from pyramid 28. An aperture 62 isprovided within plate 36 to allow plate 36 to fit over pyramid 28.Beneath plate 46 is provided filter papers 52, 54 and 64. Filter papers52, 54 and 64 are caused to move from the left to the right relative tohinge 48 by movement of handle 56 from position I to II in FIG. 8E asnoted above. Apertures 43 and 44 are provided within plate 36 tocooperate with handle 56 when it is in both position I and position IIso that device 20 can be closed during use of the device. Specifically,the exposed level of filter paper 52 is such that when the device isclosed the surface of filter paper 52 contacts the surface of testdevice 26, and absorbs liquid on that surface. Movement of handle 56(and the attached plate 66) causes a new portion of filter paper 52 tobe available for contact with test surface 26 when the device is closedagain.

This device can be manufactured using standard procedures. Specifically,once the plastic molding has been formed, filter papers 52, 54 and 64can be placed within the upper portion of the device and plate 46secured over those papers to hold them within upper portion 32.Similarly, filter paper 60 can be secured within lower portion 34 bysecuring plate 36. Both plates 36 and 46 are provided with a pluralityof small extrusions along their edges (not shown) which are adapted tomate with a lip portion 68 and 70, respectively, to hold those plates inplace. Also provided is a shelf 72 within upper portion 32 to allowplate 46 to rest on the shelf, and to allow movement of filter papers52, 54 and 64 within the inner-space 74. No such shelf is necessary inlower portion 34 since the filter material is relatively thick, and nomovement of that filter is required. Test surface 26 is readily attachedto pyramid 28 by adhesive, or other means.

Use

Referring to FIGS. 10A-I, there is shown a method by which device 20 maybe used in a method of the present invention. Specifically, in a firststep (FIG. 10A) a sample is obtained and treated in an appropriatemanner to prepare for application to the test surface. Such applicationis performed with the device open. In a second step (FIG. 10B) thesample is allowed to incubate so that any analyte present in the samplecan react with the receptor layer. At a third step (FIG. 10C) the sampleis washed from the test surface and the excess liquid allowed to flowinto the filter below the pyramid holding the test device. At this stagethe position of the upper filter material is at I. In a fourth step(FIG. 10D) the device is closed and latched so that the filter may blotthe test surface. In a fifth step (FIG. 10E) an appropriate substrate isadded, allowed to incubate (FIG. 10F) and then again rinsed (FIG. 10G)as above. At this point, the upper filter material is moved fromposition I to II, and the device again closed to allow the test surfaceto be dried (FIG. 10H). At this point, the device is again opened andthe result can be read (FIG. 10I).

Multi-test Device

Referring generally to FIGS. 9A-9E, and 11A-E, a device which willexamine a single sample for multiple analytes incorporates many of thefeatures of the single use device. The device's first position exposes anumber of test surfaces, each uniformly coated with a differentreceptive material. The device has the test protocol imprinted on theupper surface to assist the end user. Any number of test surfaces may bemounted into the device, but five independent assays are very easilyaccommodated. Sample is applied to each of the test surfaces andincubated. Following the incubation period the test surfaces are rinsedwith water. The test surfaces are mounted over a sloping trough whichwill drain the rinse solution and excess sample into an adsorbent pad inthe bottom of the device. The lid is lifted and advanced to a secondposition. This brings a blotter pad into contact with each test surfaceto dry them as described in the single use device. The lid is lifted andthe test surfaces are exposed once again. As with the single use devicethe test may be ready for interpretation at this point, or may requireadditional incubation/rinse/dry cycles. The device is easily extended toaccommodate the required number of steps. This type of device would beparticularly useful for screening patients for drugs of abuse, allergyscreening, meningitis screening, sexually transmitted diseases, TORCHpanels, and the like. The test protocol is fairly simple and would bewell suited to physician office, clinical or reference laboratorytesting. Field use would be possible when urine or whole blood is thesample to be screened. For this example it was assumed that eachreactive test surface was presented in the device as a separate 0.75 cm²test piece uniformly coated with receptive material. It is possible toapply each receptive material in discrete lines or spots across thesurface of a test piece. The test device would then approach the size ofa single use device.

It is also possible to design a device with multiple pedestals whichvery closely approximates the single use device. In this case the livinghinged lid would contain ports which are positioned precisely above eachtest surface mounted on a pedestal. The wash solution and excess samplecould be collected in an absorbent pad surrounding each pedestal orcould flow through a porous solid pedestal support to a reservoir below.

The optical substrate or support may be cut to any size desired, thus,the reactive test piece may be any size required. A uniformly coatedtest surface could be of sufficient size that a standard microtiter wellformat could be designed. The wells provide a reservoir for sampleapplication without cross contamination and exploit existing EIA assayautomation technologies. The test device could be a simple plate, of anysize, spotted with receptive material at pre-set x,y coordinates suchthat sample application is driven off of these coordinates.Cross-contamination between samples could be controlled by hydrophobicwells surrounding the reactive zones, other types of physical barriers,or by microspot sampling techniques. These types of multi-sample, singleanalyte test devices may be adapted to semi-automated or fully automatedinstrumentation, see FIGS. 12A-C. For batch testing, an instrumentedrather than eye interpretation is preferred. Batch testing surfaces maybe dried using a blotter design, a heat lamp or other such device, ormay include a forced air or nitrogen drying method. Sample residue andcontaminated rinse solution could be drained into a reservoir where itis treated prior to disposal. Or the excess sample and rinse solutioncould be drawn into a sealed section of the test device.

Batch or multi-sample devices may be designed in qualitative,semi-quantitative, or quantitative testing formats. Surfaces for batchtesting may be any size. The size will be determined by the number ofcontrols and samples to be performed in a single assay. Automated samplehandling devices and sample application devices will impact test surfacesize. Automated sample handling and batch testing applications includescreening blood for blood banks, as well as those for clinical andreference laboratories. These laboratories may require high volume,limited testing menus; high volume, large testing menus; or low volume,large testing menus. The flexibility in test surface design allows allof these requirements to be met with a single optical detection method.Additional sample handling and test device manipulation may be requiredto increase the volume of samples or the number of tests performed.

Specifically, referring to FIGS. 9A-9E, there is provided indiagrammatic representation a multi-test device of the presentinvention. This specific example is designed to test for the presence ofE. coli, Streptococcus B, Streptococcus pneumoniae, H. influenza and N.meningitidis. Generally, this device is constructed with a plurality oftest devices, namely five test devices, 100, 102, 104, 106 and 108. Thedevice has an upper slidable cover 110, a lower shelf portion 112 whichincludes a large thick filter material 114 which is removable fromsection 112 by use of a wire loop 116. Upper cover is provided withthree series of five apertures 120, 122 and 124 and with a largerectangular aperture 126. On its under surface are provided twoabsorbent wipes 128 and 130 formed of a filter material, and adhesivelybonded to the lower surface of cover 110. General indicia may also beprovided on the surface of cover as shown at 146, 148 and 150.

Also provided are a series of three cylindrical extensions extendingapproximately 4 mm from the inner surface of cover 110, labeled 132,134, 136, 138, 140 and 142. The cylindrical extensions are adapted tomate with spaces 152 provided in the lower portion of portion 112 suchthat each row of apertures in the upper cover can be specificallypositioned over the test devices or other indicia in lower portion 112,as desired. This movement is shown generally in FIG. 9B by arrows 154and 156.

Lower portion 112 is further provided with an aperture 158 located toallow excess liquid on test surfaces 100, 102, 104, 106 and 108 to drainwithin portion 112 and to be absorbed by filter 114. Lower portion 112is further provided with a series of instructions shown as 160 which arerevealed in turn as cover 110 is moved in a stepwise fashion as dictatedby the mating of cylindrical extensions 132, 134, 136, 138, 140 and 142relative to spaces 152 along slidable portion 164 so that the user ofthe device has an indication of what step is needed to perform an assayof the invention. The upper and lower portions are constructed such thatthe filter paper 128, 130 is caused to contact the surface of each testdevice at an appropriate time in the assay procedure.

Referring to FIGS. 11A-E, there is shown in diagrammatic form a methodof using the multi-assay device shown in FIGS. 9A-E (although theextension of the upper portion versus the lower portion is not shownspecifically). At step 1 (FIG. 11A), a sample is collected andappropriate reagents mixed with it. The sample is then applied to eachtest device and that device is moved one notch (i.e., one cylindricalextension is moved along arrow 156 to the next available space 152) sothat the test surface is available for such application. In step 2 (FIG.11B) the surface is again moved such that first filter material 130 isin contact with the test surfaces. Prior to this step, the test surfacesare washed and that wash solution allowed to drain through aperture 158to filter 114. After blotting, the device is again moved one notch toallow access to the test surfaces and substrate is applied (FIG. 11C).Once more, after appropriate incubation time, these surfaces are washed,with the wash solution draining to filter 114. The upper surface is thenmoved one more notch so that filter 128 contacts the test surfaces (FIG.11D). One more movement of the two surfaces relative to one anotherallows reading of the results (FIG. 11E). At each step in the process,aperture 126 indicates the step that must be taken by the user, and thusprevents incorrect use of the device.

FIGS. 12A-C are a diagrammatic representation of a three batch samplingconcepts useful in this invention. The first device FIG. 12A includes anoptically active, analyte reactive test surface #1 prepared aspreviously described. The test surface #1 is fused or glued to a plasticdevice #2 which will create individual sample wells #3. The final devicewill be configured and handled in precisely the same fashion as a 96well microtiter plate. This configuration of the test surface #1 couldbe easily adapted to any commercially available microtiter based,handling system.

The second configuration for batch testing is a device very similar tothe single use device and would be particularly useful in quick panelscreening assays (see FIG. 12B). The device in configured to include alid #10 which is hingedly attached by a hinge #7 to a bottom container#5. Bottom container #5 holds an absorbent #6 material to contain theexcess sample and wash solution. Lid #10 contains a blotter #11 which isused to dry test surface #4 in the assay protocol. The test surfaces aremounted on a pedestal #12 to facilitate the washing process. Protectivecoverings #8 and #9 hold blotter #11 and absorbent #6 in place withinthe device.

The third concept is an optically active, analyte reactive test surface#1 which contains reactive areas represented by #13 (see FIG. 12C). Thereactive area #13 may be created by spot coating, by selectiveinactivation of the receptive layer, or physical barriers betweenreactive areas. The samples are applied to the reactive areas (#13) andthen the rinse solution and excess sample flows out through the drainageport #14.

In another configuration, the test surfaces can be made as a series oflongitudinal strips with filter material on either or both longitudinaledges, and arranged to fit within a 96-well configuration.

Instrumentation

After the sample is contacted with the surface of a test device, aninstrument can be used to detect analyte binding. One such instrument isthe Sagax Ellipsometer (see, U.S. Pat. Nos. 4,332,476, 4,655,595,4,647,207 and 4,558,012, which disclosures are incorporated in fullherein and made a part hereof). Alternate instruments suited to thistechnology include traditional null ellipsometers, thin film analyzers(see FIG. 14A), profilometers, polarimeter, etc. If the interferencefilm is included in the test surface construction, then a simplereflectometer (see FIG. 14B) is adequate for quantitation.

Referring to FIG. 13, there is shown a prior art method for detectinginteraction of a light with a test surface. In the prior art, twopolarizers were provided to allow such detection. Specifically, #1corresponds to the white light source used in this prior art instrument.A standard halogen lamp is used to generate the polychromatic light. Thelight is incident on the polarizer at position #2, and is then linearlypolarized. The linearly polarized light then impinges on the referencesurface #3 which is at 70° with respect to the test surface #4. Thelinearly polarized light is reflected from the reference surface (#3) aselliptically polarized light. The light then impinges the test surface(#4) and is reflected to the second polarizer at position #5. Theinteraction of the light with test surface (#4) inverts the s- andp-components of the elliptically polarized light. The polarizers atposition #2 and #5 are matched and #5 is rotated 90° relative to #2.Light which is reflected from the test surface #4 which matches thatreflected from the reference surface #3, will pass through polarizer #5and be completely extinguished at the detector (#6). If there are anydifferences in the surface properties of surfaces #4 and #3, then someresidual ellipticity will cause an increase in intensity to be measuredat the detector #6.

Such an instrument which is useful for analysis of thin films andchanges in film characteristics is the Comparison Ellipsometer describedin U.S. Pat. Nos. 4,332,476, 4,655,595 and 4,647,207. The opticalpathway of such instruments is shown in FIG. 13, as discussed above.This instrument can use a reference surface with a wedge of thicknessesacross the surface. If thickness values are scribed onto the wedge, thethickness of a test surface may be determined relative to the wedge. Thetest surface thickness equals the wedge thickness at the point wherelight is extinguished at the detector.

The instrument operates on the basis of comparing the degree ofelliptical polarization, caused by the reflection of plane polarizedpolychromatic light, between two surfaces. Incident polychromatic lightis collimated and plane polarized. The polarized light is reflected atan oblique angle from the reference surface, which is a reflectivesubstrate with similar or identical optical characteristics to that ofthe test piece. The reflected light is then elliptically polarized as aresult of reflection. The elliptically polarized light then reflectsfrom the test surface. The test surface and reference surface arearranged perpendicular to one another such that after reflection fromthe test surface, the light is once again plane polarized where the testand reference surfaces are optically identical. If their thicknessand/or refractive indices are not identical, the light retains someelliptical character. The ellipticity is a function of the refractiveindex and the thickness differences. A second polarizer is then used tofilter the light, and removes the plane polarized light corresponding toidentical films. An increase in ellipticity will result in greater lighttransmission through the second polarizer. Thus, a change in thicknessor refractive index is transformed into a change in light intensitywhich may then be measured using conventional techniques. By employingthe Comparison Ellipsometer in this fashion, resolution to ±5 Å may beachieved. Unlike conventional ellipsometry, the Comparison Ellipsometeris designed to allow broad field measurements. This feature allowssimultaneous measurement of the entire reaction zone. Therefore,measurement errors do not arise because of non-homogeneous binding orreaction patterns.

For the applications of this invention, a more useful reference surfaceis one which is uniform. When a test surface to be analyzed has all thecomponents for colored signal generation for visual interpretation, thereference surface must also contain the optical thin film coating. Thisadditional coating is not required for the instrumented analysis. Tomaximize the signal produced by a change in thickness or mass on thetest surface, the reference standard should be approximately 50 to 100 Åthinner than the test surface, substrate, attachment layer, andreceptive material. If these two surfaces are too closely matched, thena small change in thickness or mass will result in only a small increasein intensity relative to the original background intensity. The changein intensity for small thickness changes is dramatically increased whenthe background intensity is above a certain minimum or is sufficientlybright. With this reference surface all changes in thickness or masscause a dramatic change in intensity of light measured by the detectorrelative to the test surface's initial reading. The change in intensitymay reflect an increase in thickness or a decrease depending on theapplication, see Examples 8, 12, 13, 16, and 17. The instrumentedreading protocols are given in Example 21.

For the analysis of specific binding reactions on a test surface, anumber of modifications greatly improve the performance of theComparison Ellipsometer. The original design relied on the observer'seye for inspection of the surface.

Referring to FIGS. 14A and 14B, there are shown two devices in which nopolarizers are provided, and in which a thin film can be analyzed eitherwith a single photodiode, an array, or a CCD detector array, or with areflectometer a photomultiplier detector.

The detector may be mounted where the eyepiece is located in theoriginal instrument. It may also be mounted at 90° to the side of thelight path by incorporation of a partially silvered mirror orbeamsplitter set at 45° to reflect a portion of light to a detector, andthe rest to the eyepiece for visual alignment of samples. If the mirroris inserted into the optical path, the spot intensity reaching thedetector will be only a fraction of the light available. If the detectoris directly in the optical pathway without a mirror, 100% of the sampleintensity reaches the detector. When a beamsplitter and eyepiece areincluded in the apparatus, if care is not taken, stray light can beintroduced which degrades the optical signal incident on the detector.

A photodiode array may be programmed to dedicate individual photodiodesto measure the intensity of reaction zones or spots, while otherphotodiode arrays measure the background, or control zones. Simultaneousmeasurement of the spot intensity and the background intensity allowseach reading to be accurately corrected for test surface background.

Either a linear array or a matrix array may be used. A linear array mayonly measure along one, pre-set axis of the sample spot depending on thesize and resolution available in the arrays. The matrix array couldmeasure the entire reacted spot plus background.

The instrument may also be modified to include a variable magnificationfunction or a zoom to allow different spots to fill the photodiodewithout capturing any background signal.

Specifically, two such instruments are represented diagrammatically inFIGS. 14a and 14b. The thin film analyzer (FIG. 14a) uses amonochromatic light source #1. If the light is not sufficiently linearlypolarized, then a polarizer at position #2 is used to polarize thelight. Polarizer #2 must be positioned to allow the maximum intensity oflight to pass through to the test surface #3. By off-setting the initialpolarizer a component of light polarized perpendicular to the plane ofincidence, in addition to the light polarized parallel to that plane, isallowed to interact with the surface. Light impinges the test surface #3at an angle which is sufficiently removed from Brewster's angle, between50 and 75 degrees off the normal. The polarizer/detector is set at thesame angle from the normal as the incident light source relative to thetest surface. The polarizer is set from 2° to 15° above the settingwhich aligns the polarizers for total extinction of light. Incidentangles of 30° to 40° off the normal provide adequate resolution of verydilute samples, but may not provide sufficient range for allapplications. The second polarizer, or analyzer polarizer, cannotadequately minimize the background signal when the light is incident onthe surface at angles greater than 65°. However, the dynamic range issufficient to allow for electronic reduction in the background signal.The light is reflected from the test surface #3 through thepolarizer/analyzer combination at position #4 prior to being measured atthe detector #5. The detector may be a single photodiode or a photodiodearray. A blank test surface is placed in the sample position and used toalign the second polarizer. The second polarizer should be positioned atan angle with respect to the first polarizer such that it is a fewdegrees off the minimum (maximum extinction of light through to thedetector). Thus, the background of the test surface produces a lowdetectable signal, but the change in light intensity is now a functionof the change in thickness. See Example 26.

The reflectometer (FIG. 14b) is a very simple instrument which allowsmeasurement of a color change or a change in intensity. At position #1 astandard halogen light source is used. This will provide polychromaticlight. The light source #1 is positioned relative to the test surface #2such that the maximum intensity of the incident light impinges the testsurface #2. The detector #3 may be a photomultiplier and the like. Theangle with which the light impinges the test surface #2 determines theangle at which the detector #3 is placed relative to that surface #2.

Referring to FIG. 15, in one specific example, a semi-reflective mirrorwas introduced between the zoom and the ocular at 45°. Within theocular, suitably positioned in the middle of the field and in focus wasset a reticle of an ellipse. The reticle was selected to match anaverage sample spot size. On the optical path center line, reflected 90°from the principal axis, was set a mask which matches the size of thereticle. The distance from the center of the mirror to the reticle isthe same as from the center of the mirror to the mask. The mirror wasmounted by adjusting screws so that the image seen within the reticlewould be identical to the image appearing within the mask. Behind themask, a distance of a few millimeters, was mounted a photosensitive cellarranged to only read the light which passes through the mask andtherefore from the selected image. The semi-reflective mirror is of athickness such that a secondary image appears from the second surface.This is eliminated by using a suitably coated thin mylar membrane as thebeamsplitter.

A constant light source, white light or monochromatic, is provided byusing a power supply that has feedback capabilities. A photoresistor ismounted inside the original instrument's lamp house/heat sink whichmonitors the light output of the lamp. If the light output changes acorresponding resistance change occurs, thereby affecting thecurrent/voltage sent to the lamp.

The power supply is set to deliver +15 V_(DC) to the lamp while thephotoresistor is disconnected. When the photoresistor is connected, itmaintains the light output at the level that is produced with a +15Vsource. A constant light source is required if the instrument is to beused for quantitation. The instrument may also be modified with a BNCport that will enable the output of the photodiode detector amplifier tooutput to an A/D converter board in a computer or other dedicateddevice. The dedicated device or computer reads the input signal,designates/names and stores the input, manipulates the named input,i.e., conducts statistical analyses, etc., and prints the input data andany other desired calculations derived from the input.

Specifically, FIG. 15 is a diagrammatic representation of a modificationof the prior art instrument shown in FIG. 13. A constant power source isused at position #1. The power source supplies both the white lightsource #2 and the detector #12. The white light source is a standardhalogen lamp and provides polychromatic light. As previously describedthe light passes through a polarizer at position #3 and is linearlypolarized. Polarizer #6 is matched and crossed relative to the polarizerat position #3. The reference surface #4 and the test surface #5 are aspreviously discussed. In this instrument, when light passes throughpolarizer #6 it then impinges a beam splitter at position #7. This beamsplitter splits the light such that a portion is received at thedetector #12 and a portion is received at a CCD camera at position #11.CCD #11 allows the user to locate and position the test surface #5 inthe center of the instrument's field of view. The light which is splitto the detector impinges a mask at position #9. The mask is matched tothe reticle at position #10 such that when the sample spot on the testsurface #5 is precisely centered in the reticle #10, the light whichpasses through the mask #9 to the detector #12 is reflected only fromthe sample spot. A zoom at position #8 assists in the positioning of thesample spot relative to the reticle.

The optical path used for the comparison instruments described above arelarger than desired for a number of applications. It is possible toreduce the optical path with the following modifications. Because lightemitted from a laser source (gas laser or laserdiode), is alreadycollimated and polarized, the collimating lens system can be simplifiedor eliminated. A linear polarizer is placed very close to the lightsource. This polarizer may not be necessary because the laser is oftenpolarized. The reference surface is placed at 60°-70° relative to thesample surface. The planes of incidences of the reference and samplesurfaces are orthogonal to each other. The analyzer polarizer isoriented so that maximum extinction occurs for two identical surfacesplaced at the reference and sample positions. It is important that bothpolarizers are placed with their faces perpendicular to the light beam.Any suitably small detector and electronics may be used for signalcollection, handling, and storage. For high accuracy, polarizers shouldsupply greater than 10⁵ extinction, see FIG. 16. Polarizers are builtinto the face of the light source and detector and are not labeled onthe figure.

The thin analyzer eliminates the reference surface requirements of theprevious instrument and is easier to reduce in size. The comparisonbased instruments require that a specific reference surface be designedfor each type of test surface to be used. This limits the range ofoptical substrates and optical thin films which are compatible with agiven instrument, unless means for changing the reference surface isprovided. This new instrument easily accommodates any combination ofthin film and optical substrate using a simple adjustment of theanalyzer. The instrument may provide better thickness resolution. Thisinstrument and the modified Comparison Ellipsometer may be powered witha 9V battery or other rechargeable power supply. This prototype suppliesan increase in numerical aperture, image brightness and focus. Thisallows a much higher level of magnification to be used which isimportant for work with smaller spot sizes. Samples may also be appliedmuch closer to one another than is possible with the ComparisonEllipsometer.

Specifically, FIG. 16 is a diagrammatic representation of an improvementin the prior art instrument of FIG. 13. In this case a monochromaticlight source #1 is used. A compact laser is used. A polarizer ispositioned immediately adjacent the light source at position #1. Thelens system used in the prior art instrument to supply visual inspectionof the test surface #4 in FIG. 13 is eliminated which allows a decreasein the total optical pathway to be achieved. The test surface rests andis positioned with the sample platform at position #2. Light impingesthe reference surface #3 and is elliptically polarized as discussed forthe prior art instrument of FIG. 13. The light reflects from thereference surface #3 to the test surface positioned on the sampleplatform #2. A small electronics control unit (#4) is incorporated tosupply a constant power source and to control the detector #5. A singlephotolode is used as the detector #5. The dial at position #6 is used tomove the sample platform #2 and controls the position of the testsurface. The sample platform #2 has pre-determined stops which willalign a sample spot with the detector #5. The sample spot is positionedand the detector #5 masked such that only signal from the sample spot ismeasured by the detector. A second polarizer is placed immediately infront of the detector #5.

Fluorescent Methods

A further instrumented embodiment that these optically activesubstrates, or solid supports, can address is a reflective fluorescencemethod. The fluorescence may be generated in an immunoassay, an enzymeassay, a nucleic acid assay in a homogeneous, heterogeneous,competitive, or direct format. Signal generation is not dependent on thefilm thickness in this method, however, the method does double thepathlength of the exciting incident light as well as improves thecollection efficiency at the detector (see FIG. 17C). The opticallyactive substrate, or solid support, may be any polished reflectivematerial, such as a silicon wafer.

In standard fluorescence spectroscopy, the exciting light passes oncethrough the sample, see FIG. 17a. When a reflective substrate is usedexcitation of the fluorescent species occurs at the point of incidenceand at the reflection point (17B). Generally, emission (fluorescence) isdetected 90 degrees off the axis of excitation, even though fluorescentradiation is emitted in all directions, primarily to simplify thedetector design. For example, with a point detector, excitation energyshould not be allowed to strike the detector. Gratings are frequentlyused with the excitation source, and the maximum fluorescence may not beshifted far enough to be distinguished from the excitation wavelength.This is required to reduce the effects from the high intensityexcitation source and scattering from the solution and the cell walls.With a reflective substrate, the detector and the incident light are atequal angles from the normal.

Attempts have been made to increase fluorescent assay sensitivity byincreasing the number of passes of the exciting light through thesample. This approach requires more complicated optics for focussing, ahigh output light source, and large collection optics. This method alsotends to increase the background. fluorescence from the cell andinterfering or inherent biomolecules. An increase in sensitivity mayalso be achieved by increasing the sample volume and/or the opticalpathlength. The method of the current invention provides an increase insensitivity without the complications observed with these methods.

British patent GB 2 065 298 A describes a fluorescent assay which uses areflective metal substrate generated by an evaporation process. Thecapture layer uses a specifically reactive biological particle for thebinding of analyte and then a third biological species which is bound tothe fluorescent label. The metal substrate is positioned to redirect theexciting particle toward a trap (multiple reflections) and away from thedetector.

This patent positions the photon counting system a distance from thesubstrate to allow the exciting particles to be reflected by the metalsurface toward a dark enclosure. The enclosure will absorb all thephotons that hit it which reduces the noise at the detector, while moreinduced signal from the substrate will travel to the photon countingsystem due to the reflection. The analyte is reacted in a humidincubation.

For this approach, there must be nothing in the path of the excitingphoton which starts from the light source and ends at the wall of thedark enclosure except the highly reflective metal surface and thebiological particles. With the incident angle being equal to thereflecting angle and the signal detecting system being perpendicular toand at some distance away from the substrate, the exciting particle willnot be scattered toward the detecting system.

The current invention relies on an attachment layer between thereflective substrate and the receptive, biological layer. This polymericlayer between the substrate and the biological layer does not impact thegeneration of the fluorescent signal. The attachment layer may beselected from any of the following chemicals: dendrimers, star polymers,molecular self-assembling polymers, polymeric siloxanes, and filmforming latexes. The method for production of these surfaces isdescribed in Example 5. The reflective substrate or support (opticallyactive surface) may be selected from monocrystalline silicon,glass/amorphous silicon composite, a metal, a ceramic, polycrystallinesilicon, a plastic/amorphous silicon composite, and composites of thesematerials. Methods for the production of these materials is describedabove. This method doubles the excitation pathlength without doublingthe volume of sample. In addition, coating the reflective substrate witha material anti-reflective to the exciting light eliminates the noisefrequently associated with fluorescent methods but still allowsexcitation because extinction occurs at the air/film interface only.Suitable materials include silicon nitride, silicon/silicon dioxidecomposites, silicon oxynitride, titanium dioxide, titanates, diamond,oxides of zirconium, and silicon carbide. The material and the thicknessof the material are selected to suppress light of the excitingwavelength. The exciting wavelength selected is dependent on thespecific dye (fluorophore) or label used. These materials are producedas described above. Steeper angles of incident excitation help reducethe amount of exciting light which reaches the detector.

Any number of fluorescent molecules could be utilized in thisembodiment. Fluorescent molecules such as xanthene dyes which includesfluoresceins, rhodamines, and rosamines are suited to this application.In addition the amino and carboxylic acid or isothiocyanate substitutesof these dyes are also suitable. Napthylamines such as1-dimethylaminonapthyl-5-sulfonate, 1-anilino-8-napthalene sulfonate,and 2-p-toluidinyl-6-napthelene sulfonate are also useful. Conjugationprotocols for these compounds to biological molecules are well known tothose skilled in the art. The label may be attached to a secondaryantibody, to an enzyme substrate, to a nucleic acid probe, or anysuitably selective and specific receptive material for the analyte ofinterest.

In the current invention, the optically active or reflective support,with or without the optical coating or AR film, would be coated with asuitable polymer. The polymer layer would then be coated with areceptive material specific to the analyte of interest, i.e., anantibody. The biologically reactive, reflective substrate would becontacted with a sample suspected of containing the analyte of interestand incubated for a period of time sufficient to bind the analyte to thesurface. The analyte may be mixed with the secondary receptive materiallabeled with the fluorescent material simultaneous with contact to thesurface or sequentially. In either case, the label is then immobilizedto the surface through the analyte bridge. The immobilized label isexposed to the exciting light source and the detector will measure thelevel of fluorescence. The amount of fluorescence may be measureddirectly, inversely, or indirectly related to the concentration of theanalyte of interest. Similar schemes for detection of enzyme activity ornucleic acids maybe readily derised by those skilled in the art. Thelight source and the detector can be selected from any combination ofstandard fluorescent optical components.

Analytes

Streptococcus

Group B Streptococcus (GBS), Streptococcus agalactiae, is the leadingcause of neonatal and maternal morbidity and mortality. Neonatalinfections include sepsis and meningitis, while post-partum the organismcauses endometritis, chorioamnionitis, and sepsis. For the neonate, theearly-onset disease occurs between birth and the following week. Thedisease is characterized by respiratory distress, sepsis, and shock.There are between 1.9 to 3.7 cases per 1,000 live births in the U.S.alone, with a mortality rate of 26% to 50%, and 30% of the infectedinfants developing meningitis. Of the latter group, 50% will sufferpermanent neurological damage. Infection with GBS accounts forapproximately 2,000 neonatal deaths per year and is estimated to costthe U.S. alone over $500 million per year in health care. Directcorrelation of maternal cervical/vaginal carriage of GBS and infantinfection has been demonstrated.

GBS also causes a late onset disease that occurs within the first 3months following birth. The illnesses in these cases are characterizedby central nervous system disorders, meningitis, and bacteremia. Thereis an approximately 20% mortality rate for infants with these diseases.

Maternal colonization is primarily cervical/vaginal and anorectal.Between 5% and 30% of pregnant women will be colonized with GBS.Maternal colonization may account for preterm delivery, prolonged labor,premature membrane rupture, intrapartum fever, low birth weights, aswell as the early-onset diseases. Treatment of the mother, pre-delivery,greatly improves the neonatal outcome and can eliminate the verticaltransmission of GBS. However, diagnosis of maternal GBS colonization andsubsequent treatment may not eliminate vertical transmission if thediagnosis/therapeutic program is to early in the pregnancy due tofrequent recombination of the mother. Early diagnosis does identifyneonates that may be at risk for GBS infection. But there is adocumented need for the rapid, sensitive and accurate diagnosis of GBSat the time of delivery. If such a diagnostic tool were available,prophylactic treatment of the mother could begin at the onset ofdelivery and for the infant at birth. This has been demonstrated tosignificantly reduce the risk to the neonate.

GBS consists of 5 serotypes. These are designated Ia, Ib, Ic, II, andIII. All 5 serotypes have been implicated in clinical infections. All 5serotypes contain a group specific polysaccharide which is unique, inaddition, they also contain antigens which uniquely identify theserotype. As the group specific polysaccharide identifies all theserotypes, this antigen has been the focus of immunological methods forthe identification of GBS. The "gold standard" for GBS diagnosis remainsculture identification. This process can require between 24 and 72 hoursfor accurate identification of GBS. High risk pregnancies frequentlycomplete delivery long before the culture results are available.

There are a number of different immunological tests commerciallyavailable for the detection of GBS. Clinical evaluations of thesemethods demonstrate clinical sensitivities that range from 12% to 92.3%,with an average clinical sensitivity of 50% to 60%. Analyticalsensitivities reported range from 7.6×10⁵ to 2.1×10⁷ cells. While thesemethods provide a rapid diagnosis, they do not have the requiredsensitivity to address the clinical need for timely identification ofGBS.

For example, WO 9219969, describes an assay for GBS where the solidsupport is coated with a monoclonal antibody which specificallyinteracts with one epitope of the group B specific polysaccharide, thetrirhamanose epitope. A polyclonal antibody specific to a monorhamnoseepitope is conjugated to a signal generating label. The analyticalsensitivity of the method is set at 3×10⁴ cells. Sensitivity is onlyobtained through the stringent selectivity of the capture agent. Themethod concentrates only on the dominant epitopes of GBS. The "antigencapture agent has an affinity for specifically binding to trirhamnoseepitope of GBS polysaccharide antigen" and this antigen capture agent isfurther characterized as "being able to interact with the trirhamnoseepitope in an exclusive or at least dominant fashion such that anyinteraction between this capture agent and other components of group Bstreptococcus polysaccharide antigen is at the very least low ornegligible (i.e., the interaction with the trirhamnose epitope isspecific enough for the purposes of detection and/or diagnosis of GBSpolysaccharide antigen or GBS infection)". The assay requires that theswab be placed in a receptacle coated with the antigen capture agent.The receptacle has a very high area/volume ratio. Antigen is extractedfrom the swab using an acid extraction protocol requiring 5 to 20minutes. Buffer containing Tris and Tween is then added and the swabremoved. An antigen marker agent is added and the mixture incubated foran additional 10 to 15 minutes. The receptacle is then thoroughlywashed, and a substrate added for 10 to 20 minutes. The reaction is thenstopped and the result read by spectrophotometry.

The method for-detection of GBS described in the current inventionachieves the sensitivity required in under 30 minutes. The test resultis easy to interpret and is suited to bedside or delivery room use. Theoptical test surface of this invention provides improved sensitivity dueto the unique attachment layer and works well with any group specificpolyclonal or monoclonal antibody. The antibodies do not need to exhibitdifferent epitope specificity to achieve the clinical sensitivityneeded. Antibody preparations which are a combination of varyingspecificities and affinities for the GBS antigen work well on both sidesof a sandwich format. However, antibodies with differing epitopespecificities are useful in the current invention.

Chalmydia

Chlamydia trachomatis is an obligate intracellular organism which mustbe cultured in living cells, i.e., tissue culture. Chlamydia has 15serovars and primarily causes human ocular and genital diseases such astrachoma, conjunctivitis, lymphogranuloma, venereum, non-gonococcalurethritis, and proctitis. There are approximately 3-4 million cases ofChlamydia in the U.S. annually. Only very specialized laboratories cansuccessfully culture Chlamydia but the yields are low and contaminationproblems are high. Storage conditions effect the viability of theorganism for culture. The recommended culture protocol involvesinoculating cycloheximide treated McCoy cells, a blind passage, andfluorescent staining of the inclusion bodies. Vortexing and sonicationof the sample prior to inoculation increases the positive culture yield.Sampling should include cells and mucus surrounding the sampling site.

In addition to culture techniques a number of direct immunofluorescenceand ELISA methods have been developed. These techniques provideinadequate sensitivity to detect patients with low levels of Chlamydiainfection or for individuals that are asymptomatic. An overallsensitivity of 44% relative to culture has been reported for samplescontaining less than 100 IFU, inclusion forming units. Available methodsdemonstrate a sensitivity of 82% for samples with greater than 100 IFU.

Many gram negative bacteria also produce organism specificlipopolysaccharides (LPS) similar to the one produced by Chlamydia.Detection and identification of the organism may be made based on theimmunological reactions of this antigen. Organism specificpolysaccharides may also be useful. Gram negative bacteria include butare not limited to Chlamydia psittaci, E. coli, Pseudomonasfluorescence, Azotobacter vinelandii, Aerobacter aerogenes, Neisseriagonorrhoea, Treponema pallidus, Micrococcus pyogenes, Shigella,Hydrogenomonas species, Salmonella, Hemophilus influenza, Campylobacter,Heliobacter, and Legionella.

U.S. Pat. No. 4,497,899 describes an assay based on the non-specificadsorption of antigen to a bare solid support such as silica, silicone,glass, metals, or plastic beads. There is no chemical or immunologicalbinding of the antigen to the solid support. The assay protocol involvesmixing the beads with sample which has been lysed to liberate theantigen. Following adsorption of the antigen to the beads, the beads arerinsed and transferred to a new carrier. An antibody specific to theChlamydia antigen is added and incubated for a period of time, and thenthe beads are rinsed. Another antibody specific to the anti-Chlamydiaantibody which is conjugated to the signal generating label is thenmixed with the bead and incubated, followed by a rinse step, and thenincubation with the substrate.

U.S. Pat. No. 4,497,900 describes an assay for Neisseria gonorrhoeausing a bare solid support for non-specific antigen adsorption. Thesolid support is a bare, untreated, uncoated support preferably of ahydrocarbon polymer, polystyrene, silica, silicone, glass, or metals.

U.S. Pat. No. 4,959,303 describes a method for the detection of gramnegative bacteria. The solid support used is free of any specificbinding proteins, and is essentially protein free. The solid support isa bare, hydrophobic support which may possess a positive charge. Theassay protocol requires that the antigen be mixed with an anti-Chlamydiaantibody which is non-specifically captured on the surface. An antibodyspecific to the anti-Chlamydia antibody and is conjugated to the signalgenerator is then added, followed by substrate, and then detection. Thesupport maybe any bibulous, non-porous water insoluble material.

U.S. Pat. No. 5,030,561 modifies the above approaches by using anamidine modified latex particle or polystyrene as the solid support. Theantigen adheres to the support non-specifically and the particles areused for separation of the solid phase from the liquid phase throughfiltration. The membranes used in the filtration process must be washedwith surfactant and casein prior to their use in the assay. Substratevisualization occurs at the membrane.

U. S. Pat. No. 5,047,325 details a method for the detection ofChlamydial and Gonococcal antigens using a bare or coated solid supportwhich is positively charged. Suitable solid supports include glass,cellulose, and certain polymers. The positive charge is preferably aquaternary salt which will maintain its charge over a wide pH range. Thesupport provides an additional source of non-specific antibody capturewhich must be eliminated by washing the surface with a cationicsurfactant. Samples must be pre-filtered to remove cell debris.

U.S. Pat. No. 5,075,220 uses a polymeric support with cationic surfacegroups to assist in an ionic interaction of the LPS antigen with thesolid support. The support should be free of any antibody or otherbiological compound prior to reaction with the antigen.

The Chlamydia assay of this invention uses a solid support coated with apolymeric siloxane which creates a hydrophobic surface capable ofnon-specifically capturing LPS or similar antigens. This approach willwork for identification of any LPS source. Surprisingly, it has beenfound that the best, most uniform reaction is observed when a low amountof non-specific biological material, either antibody or other protein,is coated onto the hydrophobic surface prior to antigen capture. Thisadditional coating process may also be of use in assays which use EIA,FIA, or RIA detection techniques. In the method of this invention, thenon-specific biological layer promotes uniform adhesion of theprecipitating substrate system. Any system which relies on thecombination of a solid support, particularly a hydrophobic support, witha precipitating substrate system could benefit from this observation.

RSV

Respiratory Syncytial Virus (RSV), a myxovirus, is associated withsevere lower respiratory tract illness in infants and children. Inadults, RSV usually causes a mild, afebrile, upper respiratory tractinfection. In the first six months of childhood, the organism isresponsible for 32 to 75% of all bronchiolitis and 3 to 39% ofpneumonia. These illnesses are often life threatening. The organism hasalso been associated with other types of acute febrile respiratorydiseases, such as bronchitis and pharyngitis. The organism may bedetected in nasal/pharyngeal secretions by culturing the sample prior toany freezing or exposure to elevated temperatures. The sample is used toinoculate HeLa or Hep2 cells and requires 3 to 14 days to obtain aresult. RSV outbreaks occur in late fall/early winter and latewinter/early spring and last 3 to 5 months per episode.

An early viral diagnosis allows physicians to infer patient prognosisand confirms the etiology of respiratory diseases. Three types ofdiagnostics have been used for confirmation of viral illness. First,culture isolation followed by a confirmation protocol. Second,serological assays for the detection of host response to the viralinfection, and third, direct viral antigen detection.

Culture methods for RSV involve collection of a specimen and mixing thespecimen with glass beads. Tracheal secretions and bronchoalveolarlavage specimens are routinely used. The beads are used to disrupt thespecimen cells by sonication or vortexing to disperse the virus into thetransport media. An aliquot of this is used to inoculate the cellcultures. Confirmation tests used include complement fixation andneutralization.

Serological assays rely on host response to the virus, i.e., rely on IgGproduction. However, the IgG may not be produced for two or more weeksand may persist for months to years following infection whichcomplicates diagnosis. Direct antigen detection methods are morediagnostic of active infection, but for RSV are significantly lesssensitive than culture/confirmation techniques. Direct detection methodshave included immunofluorescence, electron microscopy, ELISA, andculture.

HIV

The Human Immunodeficiency Virus (HIV) has several characteristicmarkers including gp41, gp160, gp120, p66, p24, and p18. The p24 peptideis one of 4 nucleocapsid proteins comprising the core of HIV-I and has amolecular weight of 24,000 daltons. The selective infectivity of the HIVvirus is accounted for by the p120 antigen, while gp41 is required forviral entry into the host cell. Current screening assays rely ondetection of host response, i.e., antibody production, to one or more ofthese markers. Existing immunoassay methods do not provide sufficientsensitivity for direct antigen detection.

Currently, final confirmation of HIV infection is based on Western(immunoblot) blotting techniques. These tests are costly and technicallydemanding, as well as difficult to interpret. Performance of this methodunder field conditions is usually inferior to that of referencelaboratories. Western blots are designed to detect one or more of theviral core gag proteins p17, p24, or p55; one or more of the polymeraseproteins p31, p51, or p66; and one or more of the envelope proteinsgp41, gp120, or gp160. The Red Cross requires three positive bands, onein each group. The CDC recommends at least 2 positive bands includingp24, gp41, and gp160/120, while the FDA recommends that p24, p31, gp41,and/or gp120/160 be positive to report a positive result.

The current invention provides a suitable optical platform for thedetection of host response to one or more of the HIV specific antigens.Antigens may be presented on the surface in combination or individually.

Hepatitis Virus

Clinically, the various forms of viral hepatitis are difficult todifferentiate. Therefore, serological tests are required for thediagnosis of causative agent. Five separate viruses have been associatedwith hepatitis. They are designated A, B, C, D, and E. OriginallyHepatitis C was designated non-A, non-B. Recently, a non-A, non-B likehepatitis has been postulated. Hepatitis A and E are transmitted by thefecal/oral route and cause acute infections. Hepatitis B, C, and D aretransmitted by the parenteral route and cause both acute and chronicinfections. HCV is responsible for most post-transfusion hepatitis. ManyHBV infected individuals are asymptomatic and are infective. HBVinfection has been linked with liver cirrhosis and cancer. A review ofthe serological diagnosis of hepatitis is presented in PostgraduateMedicine, volume 92, pp. 55-68, 1992.

Each form of hepatitis possesses (Ags) antigens unique to that form ofthe virus; HAV has the HAVAg; HBV has surface antigen (HBsAg), coreantigen (HBcAg), and an internal component of the nucleocapsid (HBeAg);HCV has C100, 5-1-1, C22-3 (core), C33c (core), with the N-terminal ofthe core peptide being the major antigenic region of the core antigen;HDV has the delta antigen and will frequently test positive for HBsAg;and HEV has not been well characterized. The form of hepatitis may bedetermined based on antigen detection or host antibody response. Becauseof the sensitivity required for direct antigen detection, currentdiagnostic assays detect antibody response to specific antigen/s. Hostresponse may produce IgM and/or IgG and differentiation of the antibodyresponse may be required to establish the active state of infection. Forexample, IgM anti-HAV indicates an acute infection, while IgG anti-HAVindicates previous infection.

The current invention provides a useful platform detection technologyfor diagnosis of hepatitis. A combination of antigens can be immobilizedon the test surface to detect host response to those antigens. Thesurface panel can detect a combination of antigens specific to onehepatitis virus, e.g., HBV, where the surface can be coated with one ormore of the following: surface antigen, core antigen, or the e antigen.Or the panel can be coated with one or more antigens which discriminatebetween HAV, HBV, HCV, HDV, and HEV using a single sample. Such ascreening test can be developed in either the visual, qualitative formator in a fully automated, instrumented format. Antibody detection can bespecifically tailored for IgG, IgM, or both.

The following examples illustrate various procedures by which testdevices of this invention can be optimized. They also provide examplesof particularly useful combinations of each surface layer describedabove for use with instruments or eye-read results. Those in the artwill recognize that these procedures can be used to optimize equivalenttest devices to produce those useful in this invention.

EXAMPLE 1

Diffuse Surface

Silicon wafers lapped with varying particle sizes to produce varyinglevels of diffuse refection, were coated with silicon nitride to athickness of 500 Å and refractive index of 2.0 to produce an AR wafer.This produces a gold interference color. The wafers were initiallyinspected for the amount of reflectivity observed, or for the remainingspecular characteristics. These wafers were then coated with anaminosilane as described below, and then antibody coated with ananti-Strep A polyclonal antibody.

Test surfaces were chemically activated by application ofN-(2-aminoethyl)-3-aminopropyltrimethoxysilane by the followingprocedure:

1. The AR wafers were oxygen plasma etched for five minutes in a vacuumat 0.7 Torr oxygen pressure with a plate current of 175 D.C.milliamperes and 250 RF watts.

2. The wafers were placed in a quartz rack and inserted into a vacuumdesiccator with a vessel containing 5 microliters ofN-(2-aminoethyl)-3-aminopropyltrimethoxysilane. The vacuum was evacuatedto 0.06 Torr for 30 minutes. The temperature of the desiccator wasraised to 100 degrees over the course of one hour to complete the vaporphase deposition of the aminosilane.

3. 20 micrograms/ml of a polyclonal anti-Strep A preparation in 20 mlsof PBS (phosphate buffered saline), 10 mM potassium phosphate, 0.8%NaCl, at pH 7.2), and 1% by volume glutaraldehyde were combined to formthe receptive material solution. The wafers were placed in a petri dishand the receptive material solution was added.

4. The wafers were incubated at room temperature (about 20° C.) in anagitation bath for 15 hours.

5. Following incubation, the wafers were rinsed with deionized water toremove the unbound antibody.

6. The surface was incubated in a stabilizing solution and incubated forone hour in an agitation bath. The stabilizing solution was made of 2ug/ml acid hydrolyzed casein, 1% (v/v) glycerol and 2% (w/v) sucrose inPBS.

7. Following the stabilizing process, the surface was rinsed withdeionized water and then dried under a stream of nitrogen.

The wafers were then reacted with samples containing varying levels ofStrep Group A antigen and a latex secondary reagent, described inExamples 14 and 15, by incubation for 2 minutes at room temperature. Theslides were rinsed with deionized water and dried under a stream ofnitrogen. No difference was observed in the amount of silaneincorporated or antibody attached.

The results are shown in Tables 1 and 2, and demonstrate that suitablynon-specular surfaces allow viewing at any angle and with a highersensitivity than a specular surface.

                  TABLE 1    ______________________________________    Lap      Average   Dek-tak ®                                Dek-tak ®    Particle Size             Particle Size                       Separation                                RMS    Comments    ______________________________________     10-20 micron             15 micron 20 micron                                2995   Diffuse, no angle                                       dependence,                                       color clear at all                                       viewing angles     20-40 micron             30 micron N/A      N/A    Diffuse, minimal                                       specular                                       character, low                                       angle                                       dependence,                                       color constant at                                       most viewing                                       angles     40-60 micron             50 micron N/A      N/A    Less diffuse,                                       more specular                                       character, more                                       viewing angle                                       dependence,                                       color starting to                                       show angle                                       dependence    80-100 micron             90 micron 40 micron                                4779   Slightly diffuse,                                       very specular,                                       strong angle                                       dependence,                                       color varies with                                       viewing    ______________________________________

                  TABLE 2    ______________________________________    Visual Interpretation of a Strep A Assay on Different    Lapped Wafer Surfaces    Concentration of Strep Group A Antigen    Wafer 0.000  0.008  0.016                             0.032                                  0.064                                       0.128                                            0.250                                                 0.500                                                      1.000    ______________________________________    15    --     Trace  +    ++   ++   +++  +++  ++++ ++++    micron    30    --     --     Trace                             +    ++   ++   ++   +++  +++    micron    50    --     --     Trace                             Trace                                  +    ++   ++   ++   +++    micron    90    --     --     --   --   --   +    +    ++   +++    micron    ______________________________________

EXAMPLE 2

Glass Substrate

A four inch diameter sodium borosilicate glass was coated with a thinfilm, 10-50 Å, of aluminum (chromium can also be used) to effectivelyincrease the reflectivity of the glass and block back surfacereflections. This material was then coated with amorphous silicon by athermal deposition process, as described above, and then with a layer ofsilicon nitride, approximately 500 ±3% Å in thickness.

The surface was prepared for antibody coating as described in Example 11below. In this example, a polyclonal antibody to Streptococcus Group A(GAS) was used to coat the surface. Specifically, 100 μl samples ofdiluted or undiluted GAS antigen was mixed with 50 μl anti-GAS surfaceactivator particle, and assayed as described in Example 11. A comparisonof this surface to a silicon nitride coating on a silicon wafer usingthe GAS model assay was conducted. The results are shown in Table 3 anddemonstrate that glass provides as useful a substrate as does silicon.

                  TABLE 3    ______________________________________    FOLD            GLASS      SILICON    ANTIGEN DILUTION                    SUBSTRATE  SUBSTRATE    ______________________________________    0.0             -          -     1:256          +/-        -     1:128          +          +     1:64           +          +     1:32           ++         ++     1:16           ++         ++    1:8             +++        +++    1:4             +++        +++    1:2             +++        +++    DIRECT          +++        +++    ______________________________________

EXAMPLE 3

Construction of an AR Test Surface

An optical substrate, for this example monocrystalline silicon, wascoated with a thicker layer (for example 750 to 800 Å) of material suchas silicon nitride which approximates the square root dependencerequired for an AR film. Thickness was then selectively removed by achemical etching technique, described above, creating approximately 50 Åsteps across the substrate. This process produces a wedge ofinterference colors across the surface of the substrate (see FIG. 3).The substrate was then coated with the attachment layer and receptivematerial. Assays were performed using negative, low positive, andintermediate positive samples.

Any combination of attachment layer, receptive material, and assayprotocol may be used in this analysis. The specific example used a wedgeof silicon nitride on silicon which was coated with T-structuresiloxane, as described in Example 5 below. The siloxane coated AR filmwas coated with a polyclonal anti-Strep A antibody as described inExample 11. The assay protocol followed was as described in Example 19.Samples were placed in the center of each wedge (different thickness).After completion of this assay the entire substrate was examined for theselection of the wedge or wedges which provide: 1) the cleanest negativeresponse --the least detectable non-specific binding, 2) the bestsensitivity, and 3) the best visual contrast. Once a 50 Å range ofthicknesses was selected, greater resolution can be achieved byuniformly coating a substrate to the maximum thickness selected in thefirst experiment (for example 550 Å), and then etching this surface in10 Å steps. This method rapidly allows the selection of the requiredoptical thickness which provides the best "apparent" color change incombination with the biological materials.

EXAMPLE 4

Preparation of TiO₂ Optical Coating

All measurements are based on the volume of material used. Theorganotitanates may be purchased from Dupont, of particular utility isTyzor TPT (tetra isopropyltitanate) but tetra n-butyltitanate may besubstituted. One ml of TPT was mixed with three mls of glacial aceticacid, 3 mls alcohol, 3 mls deionized water, and 10 μl of 3M's FC171fluorosurfactant. Isopropanol, t-amyl alcohol, ethanol or acetone may beused with water for this application. Ethanol should be avoided as itleads to precipitation of the titanium.

Three to five hundred microliters of this mixture was applied to anoptical substrate and a uniform film produced by a static spin coatingtechnique. The film thickness should be 495 Å±15 Å. The film was curedto the substrate by heating it to 250° C. for two hours or microwavingat 400 watts of power for two minutes. The optical substrate used inthis example was a monocrystalline silicon wafer. The acceptabletemperature limits will also be dependent on the type of opticalsubstrate used. Plastic will not tolerate the 250° C. cure, but glasswill. The curing conditions selected here generate a film with arefractive index of 2.0 (±3%) which is adequate for this application.

Caution must be used to ensure that the optical substrate is clean,i.e., particle free, and that the coating solution is particle free.Particulates introduce coating defects in the film during the spincoating application process.

EXAMPLE 5

Production of Attachment Layers

The designations given here for various attachment layer materials willbe used throughout. Attachment layer materials:

#1: PEI-(Trimethoxysilylpropyl)polyethyleneimine

#2: PEI/DMDCS-PEI+DimethylDichlorosilane

#3: Polystyrene

#4: MSA-Starburst; 5th generation

#5: T-Polymer-Aminoalkyl T-structure branch point polydimethyl siloxane

#6: TC7A-film forming latex

#7: DMDPS-Dimethyldiphenyl siloxane copolymer

#8: Mercapto-Mercaptopropylmethyldimethyl-siloxane copolymer

#9: BAS-N-(2-Aminoethyl-3-aminopropyl)-trimethoxysilane

#10: PBD-Triethoxysilyl modified polybutadiene

#11: PAPDS-(methylphenyl)methyldodecylmethylaminopropyl-methyl siloxane

These chemicals were used to form attachment layers as follows:

#1: PEI (Petrarch; Bristol. Pa.)

A 1:500 dilution of the stock silane was made in methanol. A 300microliter sample of this solution was placed on a 100 mm virgin testsilicon wafer by micropipette, although automated aerosol or spraydelivery systems are equally useful, while the wafer was spinning at7,000 rpm on a photoresist spin-coater. Spin coating can rapidly processa large number of substrates and is readily automated. While spincoating is detailed here, it is not the intention to limit thisinvention to this type of attachment layer production. Alternatesolution based or vacuum based (where appropriate) depositions could beeasily designed by those skilled in the art. PEI coated substrates werecured at 100° C. under 0.1 mm Hg for 60 minutes. A final attachmentlayer of 80 Å as measured by conventional ellipsometry is generallypreferred, but other thicknesses have been utilized.

#2: PEI/DMDCS; DMDCS (Sigma Chemical Co., St. Louis, Mo.)

A PEI coated substrate may be further processed by treatment with DMDCS.This creates branch points along the linear PEI chain and causes thesurface to perform more as a T-polymer coated surface. A 100 milliliterstock of 2% DMDCS was prepared in 1,1,1-trichloroethane (v/v). The PEIcoated substrate was submerged in the solution for 60 minutes at 25° C.The substrate was removed from the DMDCS coating solution and rinsedwith 95% ethanol and finally dried under a stream of nitrogen. A finalattachment layer of 200 Å as measured by conventional ellipsometry isgenerally preferred, however other thicknesses are possible.

#3: Polystyrene (Becton Dickinson, Oxnard, Calif.)

Approximately 0.05 g of a polystyrene was dissolved in 2 milliliters oftoluene. A solution was applied by the spin coating technique describedabove. Substrates were cured for 60 minutes at 25° C. prior toutilization. A final attachment layer of 200 Å is generally preferred,however other thicknesses are possible.

#4: MSA-Starburst polymers (Polysciences, Warrington, Pa.)

A 1:4 dilution of the 5th generation Starburst (0.5% solids) wasprepared in methanol. A 200 microliter sample of this solution wasapplied to the substrate using the spin coating method at a spin rate of3500 rpm. This attachment layer was cured for 120 minutes at 25° C. Afinal layer of 40 Å is generally preferred, however other thicknessesare possible.

#5: T-Polymer (Petrarch, Bristol. Pa.)

A 1:300 (v/v) dilution of the T-polymer was prepared in2-methyl-2-butanol. The attachment layer was applied to the substrate bythe spin coating method and was cured for 24 hours at 140° C. prior touse. A final layer of 100-160 Å is generally preferred.

#6: TC7A (Seradyn, Indianapolis, Ind.)

The 30% stock solution was diluted to a 0.5% solid in methanol. A 300microliter sample is applied to the substrate using the spin coatingtechnique and is cured at 37° C. for 120 minutes prior to use. A finalthickness of this material is preferred to be 240 Å.

#7: DMDPS (Petrarch)

A 1:100 (v/v) stock solution of the siloxane in toluene was prepared andapplied utilizing the spin coating technique and curing protocoldescribed for the T-polymer. A preferred final thickness is 200 Å.

#8: Mercapto (Petrarch)

A 1:300 (v/v) stock solution of the siloxane was prepared in toluene.The coating and curing protocol were as described for PEI. A preferredfinal thickness is 200 Å.

#9: BAS (Petrarch)

A 1:100 (v/v) solution of silane was prepared in toluene. A 200microliter sample was used in the spin coating protocol. The wafer wascured for 2 hours under 0.1 mm Hg at 140° C. A preferred final thicknessis 30 Å.

#10: PBD (Petrarch)

A 27.5 microliter volume of the stock silane was mixed with 3275microliters of toluene. The spin coating volume was 300 microliters ofthis mixture and wafers were cured for 60 minutes at 120° C. A preferredfinal thickness is 100 Å.

#11: PAPDS (Petrarch)

A spin coating volume of 200 microliters of 1:100 (v/v) of siloxane intoluene was used and wafers were cured for 120 minutes at 100° C. priorto use. A preferred final thickness is 200 Å.

The above-noted concentrations, volumes, weights, spin coating speed,buffers, incubation time and conditions, and all other reagents orprocesses described throughout these examples are intended to describepreferred embodiments only, and are not limiting in this invention.

EXAMPLE 6

Comparison of Attachment Layer Materials For Antigens

A system was designed for the analysis of attachment layer materialefficiency in attaching an antibody as receptive material to amonocrystalline silicon substrate. This procedure can be used foroptimization of other systems of this invention. Achieving a dense,reactive layer of antibody has been demonstrated to be more difficultthan other layers of receptive materials due to more stringentorientation requirements. An ELISA system was designed for evaluation ofan attachment layer. A monoclonal anti-horseradish peroxidase (HRP) wasbound to an attachment layer as the test receptive material, thenvarying levels of horseradish peroxidase (HRP) were placed on thesurface to produce a standard curve. Microtiter wells were antibodycoated under the same conditions as a control.

All surfaces were antibody coated from a solution of 0.05M PBS, pH 7.4containing 20 μg/ml of the monoclonal anti-HRP (Sigma Chemical Co., St.Louis, Mo.) for 16 hours at 25° C. The coated substrates were submergedin the coating solution. Peroxidase (Sigma Chemical Co., St. Louis, Mo.)concentrations were allowed to react with the test surface or themicrotiter wells for 30 minutes at 37° C. and then unbound peroxidasewas removed by rinsing with deionized water. TMB (Kirkegaard and Perry)substrate was then added to all test surfaces and allowed to react for 2minutes at 25° C. for color development. Fluid from each spot on thetest surface was transferred to an uncoated microtiter well containingstopping reagent and the optical density at 450 nm recorded. Stoppingreagent was added directly to the microtiter wells of the comparisonplate and it was similarly read.

The results of this study are presented in Table 4. Surfaces wereevaluated in terms of sensitivity (resolution of low concentrationsrelative to the negative control) and dynamic range. For controlpurposes each attachment layer was also coated with rabbit IgG and thenevaluated in the peroxidase assay. Insignificant interaction ofperoxidase with all rabbit IgG coated attachment materials was observed.The raw silicon substrate was also examined under similar conditions,and found to exhibit very little active receptive material binding tothe surface. (Data is reported as optical density measured at 450 nm.)

                                      TABLE 4    __________________________________________________________________________    PEROXIDASE CONCENTRATION    (ng/ml)    Test Surface           0.0 15.6                   31.25                       62.5                           125.0                               250.0                                   500.0                                       1000.0    __________________________________________________________________________    Nunc*  0.005               0.634                   0.646                       0.863                           0.876                               1.252                                   1.561                                       1.413    Dynatech*           0.017               0.161                   0.150                       0.279                           0.662                               1.173                                   1.465                                       1.598    PEI/DMDCS           0.007               0.136                   0.264                       0.371                           0.428                               0.714                                   1.118                                       1.493    T Polymer.sup.a           0.030               0.076                   0.107                       0.111                           0.276                               0.498                                   0.730                                       0.850    T Polymer.sup.b           0.015               0.137                   0.328                       0.365                           0.473                               0.682                                   0.946                                       0.810    MSA    0.003               0.037                   0.100                       0.166                           0.305                               0.373                                   0.511                                       0.428    PEI.sup.d           0.008               0.175                   0.238                       0.636                           0.651                               0.702                                   0.817                                       0.743    TC7.sup.c           0.016               0.065                   0.109                       0.159                           0.179                               0.399                                   0.324                                       0.215    MERCAPTO           0.000               0.259                   0.514                       0.658                           0.881                               0.957                                   1.143                                       1.558    DMDPS  0.015               0.039                   0.036                       0.166                           0.100                               0.152                                   0.259                                       0.442    Polystyrene           0.000               0.248                   0.343                       0.444                           0.631                               0.756                                   0.795                                       0.878    BAS.sup.d           0.002               0.008                   0.012                       0.026                           0.055                               0.100                                   0.120                                       0.210    PBD    0.011               0.013                   0.047                       0.041                           0.072                               0.108                                   0.124                                       0.143    PAPDS  0.004               0.314                   0.559                       0.515                           0.790                               0.822                                   1.259                                       1.186    __________________________________________________________________________     .sup.a TPolymer was applied to the substrate to a final thickness of     240Å.     .sup.b TPolymer was applied to the substrate to a final thickness of     55Å.     .sup.c TC7A was applied to a final thickness of 246Å.     *Microtiter wells from these suppliers were used for comparison to the     optical test surfaces.

This study clearly demonstrates the utility of a siloxane as anattachment layer on a thin film substrate relative to treating suchsubstrates with PEI or BAS. There is also variability in the utility ofthe individual siloxanes, suggesting the functional groups of thesiloxane may influence the reactivity of the receptive material. Themolecular self-assembling polymers also show enhanced performance as anattachment layer relative to BAS, but are not as useful as the siloxanematerials. While the TC7A surface activator performed poorly in thisassay system it has marked utility in subsequent examples.

EXAMPLE 7

Comparison of Attachment Layer Materials For Antibodies

For this analysis, varying attachment layers were coated by immersion ina solution of 20 μ/ml of rabbit IgG (Sigma Chemical Co., St. Louis, Mo.)in 0.05M PBS, pH 7.4 for 16 hours at 25° C. Different levels of HRPlabeled goat anti-(whole molecule) rabbit IgG antibody (Sigma ChemicalCo., St. Louis, Mo.) were allowed to incubate with the test surface for15 minutes at 37° C. Unbound material was removed by rinsing withdeionized water. The TMB substrate solution was applied to the surfaceand allowed to react for 2 minutes at 25° C., and then the solutiontransferred to an uncoated microtiter well, containing stoppingsolution. (The optical density of these samples was measured at 450 nm.)The results are shown in Table 5.

                                      TABLE 5    __________________________________________________________________________    Goat Anti-Rabbit-HRP Concentration    (ng/ml)    Test Surface           0.0 15.6                   31.25                       62.5                           125.0                               250.0                                   500.0                                       1000.0    __________________________________________________________________________    PEI/DMDCS           0.010               0.035                   0.085                       0.123                           0.290                               0.289                                   0.469                                       0.572    T Polymer.sup.a           0.015               0.060                   0.061                       0.136                           0.424                               0.437                                   0.585                                       0.715    MSA    0.073               0.019                   0.033                       0.085                           0.153                               0.227                                   0.616                                       0.799    Raw Silicon.sup.b           0.000               0.000                   0.001                       0.012                           0.026                               0.037                                   0.128                                       0.280    __________________________________________________________________________     .sup.a TPolymer was applied to the substrate to a final thickness of     53Å.     .sup.b Raw silicon is substrate material alone

In this study the various test surfaces were used to demonstrate theutility of the system in the detection of an antibody capture. In thiscase the siloxane attachment layer and the molecular self-assemblingattachment layer performed equally well. The substrate without theaddition of an attachment layer demonstrated very little available orreactive receptive material, demonstrating the need for an attachmentlayer.

EXAMPLE 8

Competitive Assay Format

DNP is a small molecule, not atypical of the molecular range exhibitedby therapeutic drugs, drugs of abuse, pesticide residues, or organicresidues. It is often desirable to assay small molecules such as theseusing a competitive assay format.

This example describes the method and results of an assay quantitatingthe concentration of DNP in the sample. The surface was coated witheither the hapten or a carrier which was conjugated with the hapten. Thehapten may be directly immobilized to the surface if appropriatechemistries are available, or passively attached to the surface. Thesame options apply to the hapten/carrier conjugate.

Test samples were mixed with a material which will react not only withthe free hapten in the test sample, but also with the immobilizedhapten. One of the most commonly used reactive materials is an antibodyspecific to the hapten. Any highly specific binding reagent could besubstituted for the antibody. The extent of capture of this reagent isinversely proportional to the concentration of free hapten in theoriginal test sample. The test may be designed to produce quantitativeor qualitative results.

Materials and reagents were prepared as follows:

1. Monocrystalline silicon wafers, 4 inches in diameter, n=4.02, virgintest quality, polished on one side, 1-0-0 crystal orientation, werecoated with silicon monoxide to a final thickness of 550 (±10) Å and arefractive index of 2.0 (±0.05). The thin film interference colorproduced by this material is gold. The silicon monoxide was applied tothe wafers by a standard chemical vapor deposition technique.

2. The wafers were activated with the bis-aminosilane vapor coatingprocess described in Example 1.

3. These amine derivatized surfaces were placed in 30 mls of solutioncontaining phosphate buffered saline (PBS), pH=7.2 and 5 mg/ml DNPconjugated to human serum albumin (HSA) (DNP-HSA) in a Falcon 100 mmTissue Culture Dish. Wafers were coated at 37° C. (±2° C.), 98% humidityuntil 40 Å of DNP-HSA was deposited on the surface (approximately 30minutes). A Gaertner Ellipsometer was used for all thicknessdeterminations. The wafers were removed from the coating solution,rinsed with deionized water, and dried under a stream of nitrogen.

4. Goat anti-DNP was mixed in PBS to a concentration of 1.2 mg/ml. DNPwas dissolved into water. Antibody and DNP were mixed 1:1. A 20 μlsample of the mixture was applied to the coated surface and incubatedfor 10 minutes at room temperature. Unbound materials were rinsed fromthe surface with deionized water and the surface dried under a stream ofnitrogen. The slide was visually examined and, in addition, the changein mass at the surface measured by a change in light intensity using amodified Sagax Comparison Ellipsometer, see FIG. 15. Reading protocol isgiven in Example 21. Visual examination gives a semi-quantitativeestimation of the concentration when a sample is compared to a standardcurve.

The results are shown in Table 6.

                  TABLE 6    ______________________________________    Concentration of                   Relative    DNP Added in ng/ml                   Ellipsometric Intensity                                 Visual    ______________________________________    0.0            53.2          ++    0.031          40.4          +    0.062          34.0          +    0.125          27.4          +/-    0.500          26.6          +/-    1.000          23.3          +/-    100.0          15.0          -    ______________________________________

EXAMPLE 9

Small Molecule Detection

A 4-inch monocrystalline silicon wafer with an index of refraction of4.02 at a specified wavelength was coated with silicon oxynitride. Therefractive index of this coating of silicon oxynitride was 1.98 with athickness of approximately 540 Å.

The wafer was then chemically activated by application of approximately50±2 Å of (N-trimethoxysilylpropyl) polyethyleneimine (Petrach Systems,Bristol, Pa.) using standard wafer spin coating techniques designed forapplication of photoresist. The wafer was cured in an oven at 140° C.for a period of 2±0.1 hours.

A fresh 1% solution of a small analyte, trinitrobenzene sulfonic acid(TNBS), was prepared in deionized water. A 25 μl drop of deionized water(control) and of the TNBS solution was placed on the surface of theamine coated wafer. The drops were allowed to react with the surface forfive seconds. The surface was rinsed with water and dried with a streamof pressurized air. Upon visual inspection under polychromatic light thearea contacted with the TNBS is a red-purple color.

The change in thickness on the surface caused by TNBS binding isapproximately 20 Å. This was sufficient to produce a visuallydiscernable color change in the area where the TNBS bound.

EXAMPLE 10

Enzyme Detection

A 4-inch monocrystalline silicon wafer was coated with an AR film of525±3% Å silicon nitride (refractive index 1.97±0.05).

The deep gold colored wafer was coated with approximately 100±2 Å ofaminoalkyl-(t-structured)-polysiloxane (Petrarch Systems, Bristol, Pa.).The gold colored wafer was cured for two hours at 140° C. The wafertakes on a very slight purple tint.

The siloxane coated wafer was placed in a phosphate buffer containingacid soluble collagen. The collagen solution was prepared in thefollowing manner. Acid soluble collagen type I from calf skin (SigmaChemical, St. Louis, Mo.) was dissolved in 1 molar acetic acid adjustedto pH 4 at a concentration of 5 mg/ml. Then, a 0.1 molar phosphatebuffered saline, pH 6.8, containing 20 ug/ml of the acid dissolvedcollagen was prepared. This solution was used to coat the wafer for aperiod of two hours at room temperature. Thirty mls of solution wereplaced in a Falcon Tissue Culture Dish and the wafer submerged in thesolution. The wafer was rinsed with water and dried with a stream ofpressurized air. The wafer had a dark purple/blue color. Solutions ofcollagenase enzyme (Boehringer-Mannheim) in a 0.1 molar Tris-Hcl buffer,pH 7.2, containing 50-mM calcium ion were prepared with 0 to 1 units ofactivity per ml of solution to evaluate the collagen coated wafer. Oneunit is equal to the amount of enzyme that hydrolyses 1 μmole of FALGPAper minute at 25° C. In this system the enzyme will degrade the collagenon the wafer surface and cause a thickness decrease. The thicknessdecrease is opposite of the other examples described in this invention,which involve color change due to thickness increases.

A 25 μl drop of collagenase enzyme at a concentration of 0.5 units/mlwas allowed to react with the collagen coated wafer for five minutes.The wafer was washed with deionized water and dried with a pressurizedair stream. Upon visual inspection areas contacted with the enzyme had agold appearance while the background remained dark red to purple. Theresults are shown in Table 7.

                  TABLE 7    ______________________________________    Collagenase Concentration                    Visual       Color    ______________________________________    0.0 μ/ml     -            purple/blue    0.1 μ/ml     +            purple    0.2 μ/ml     +            purple    0.5 μ/ml     ++           pale purple    0.8 μ/ml     ++           pale purple      1 μ/ml     +++          gold    ______________________________________

EXAMPLE 11

Attachment Layer Evaluation

A silicon substrate was prepared by processing diamond sawed wafers froma monocrystalline silicon ingot in a series of steps known to thoseskilled in the art as lapping. Sawed wafers were lapped with an abrasivematerial, etched to a more uniform surface profile with acid or causticsolutions, then further lapped to a progressively finer level of surfaceroughness. For this application, an abrasive preparation of 12-21 micronaluminum oxide particles with a mean size of 15 microns was used toproduce a diffusely reflective substrate. For this particular study, thesubstrate prepared as described above was coated with silicon nitride toa final thickness of 550 Å. While this is the combination of materialsdescribed, any AR material at varying thicknesses may be used withinthis invention. The test surface was then treated with a number of theattachment layer materials as described in Example 5.

These test surfaces were coated in solution with 20 μg/ml of a rabbitanti-Streptococcus Group A (Strep A) antibody in 0.1M HEPES, pH 6.0 for60 minutes at 25° C. The test surfaces were reacted by placing a 10microliter spot of a control solution either free of or containing StrepA antigen and a latex mass enhancing reagent (see, Example 14) andincubating for 2 minutes at room temperature. Test surfaces were thenrinsed with deionized water and dried under a stream of nitrogen.

The negative control was prepared by mixing 1 part 2M NaNO₂ with 1 part2M acetic acid and neutralizing with 0.66N NaOH. The positive control, acommercially available buffer-based preparation of extracted antigenfrom cultured Strep A cells was diluted in the extraction media prior touse. Samples were mixed 1:2 with a secondary latex reagent prior toapplication to the test surface. Results in Table 8 are reported as thehighest dilution of positive control capable of being visualized above anegative control.

                  TABLE 8    ______________________________________    Test Surface                Highest Detectable Dilution    ______________________________________    T-Polymer    1:256    TC7A        1:8    BAS         No visual response    PEI         No visual response    PEI/DMDCS   1:16    DPhDMS      No visual response    MSA         1:64    ______________________________________

This study was designed to demonstrate the utility of the attachmentlayers in an antigen capture assay where the result is a visual signalon a diffusely reflecting substrate. In this case BAS and PEI,demonstrate little functional receptive material binding. The varioussiloxanes demonstrate varying ability to adhere the receptive material.The best assay performance is obtained with the T-polymer siloxane. Boththe molecular self-assembling attachment layer and the surfaceactivator, TC7A, show some utility in this assay system.

EXAMPLE 12

Instrumented Assay

The monocrystalline silicon substrate used in this example is a polishedwafer surface. The attachment layers were applied as in Example 5 andantibody was applied as in Example 11. The assay was conducted asdescribed in Example 11. The positive control used contained a dilutionof the Strep Group A antigen. Once dried, the reacted test surfaces wereexamined with the Sagax Comparison Ellipsometer and the photometricanalysis of the reflected light intensity was recorded in terms of amillivolt reading (see, Table 9).

                  TABLE 9    ______________________________________    Test Surface               Negative Control (mV)                             Positive Control (mV)    ______________________________________    PEI        36.0          133.0    PBD        21.2          37.7    TC7A       0.0           56.0    T-Polymer  15.5          286.4    MSA        0.0           136.0    ______________________________________

All attachment materials tested are useful in this assay, with PEI, MSA,and T-polymer providing optimum results.

EXAMPLE 13

Collagenase Activity

A TC7A test surface was prepared as in Example 5, the monocrystallinesilicon wafer was coated directly. The test surface was submerged in asolution of 0.1M Tris-HCl, pH 9.0, containing 4.9 ug/ml of humancollagen Type 1 (Sigma Chemical Co., St. Louis, Mo.). The test surfacewas coated for 60 minutes at 25° C. Test surfaces were rinsed withdeionized water and dried under a stream of nitrogen prior to use. A 143Å layer of immobilized collagen was produced. Varying dilutions ofcollagenase (Boehringer-Mannheim, Indianapolis, Ind.) were prepared in abuffer containing 0.005M CaCl₂ and 0.1M Tris-HCl, pH 7.6. Fivemicroliter spots of the varying concentrations of collagenase wereapplied to the test surface and incubated for 5 minutes-at roomtemperature.

Reacted surfaces were rinsed with deionized water and dried under astream of nitrogen. Reacted surfaces were examined with the SagaxComparison Ellipsometer and reflected light intensity recorded. In thisexample the receptive material layer is degraded by the collagenase andholes are produced in the receptive material to give a progressivelymore negative signal as a function of increasing activity orconcentration of collagenase. Collagenase activity is reported inunits×10³ /ml. Activity was measured as light intensity in millivolts(see, Table 10).

                  TABLE 10    ______________________________________    Collagenase            Run #1   Run #2  Run #3                                   Average                                         S.D.  % CV    ______________________________________    0.0     -15      -1       2    -4.7  9.0   200.0    100.0    60      55       68   61.0  9.6   10.8    200.0   125      93      108   108.7 16.0  14.7    300.0   181      118     188   162.3 38.6  23.4    500.0   271      228     228   240.0 27.1  11.3    ______________________________________

This study was designed to demonstrate the production of a test surfacefor the detection of an enzyme activity. While the demonstrated activityis degradative in this case, the production of a system to measureenzyme activity in terms of a synthetic activity can also be envisioned.In this case, the TC7A attachment layer demonstrated nearly a 3 foldincrease in acceptance of receptive material relative to the T-polymersiloxane (results not shown).

EXAMPLE 14

Mass Enhancement

This example demonstrates use of mass-labelled antibody and selection ofan appropriate mass-providing reagent. Such selection can be used fordetermination of optimum mass labels for other systems of thisinvention.

Surface Activator particles were purchased from Bangs Laboratories,Carmel, In.; Amide Particles: Lot Numbers L910108A (SA7-015/758) orL901015J (B7-015FF/181); or Carboxylate Particles: Lot Number L9004108(SA1-015/787). TC3, TC3X, TC7, and TC7X are similar film-formingparticles which were purchased from Seradyn, Inc, Indianapolis, Ind. Allof the TC designated preparations were carboxylic acid containingstyrene-butadiene copolymers. Of these preparations only the TC7particles were extensively examined. The TC3 preparations produce a veryopalescent film and were not used. Seradyn particles examined were TC-7AProduct Number CML, Lot Number 1K30; TC-7X Lot Number 1M92, TC-7 LotNumber 1V18 (F040690), TC-3X Lot Number 1R35, and TC-3 Lot Number 1J44.The surface activator preparations offer more flexibility in thechemistries of immobilization as both carboxylate and amide particlesare available. The amide particles are readily converted to thehydrazide particle as described in U.S. Pat. No. 4,421,896.

In this study, monocrystalline silicon, virgin test wafers were useddirectly. These wafers were coated with a T-polymer siloxane (PetrarchSystems, Bristol, Pa., Catalog Number PS401, Lot Number) by applicationof 300 microliters of 1:300 (v/v) dilution in 2-methyl-2-butanol of thestock siloxane using a spin coating device. The T-polymer was cured tothe surface of the wafer by a heat treatment for 120 minutes at 120° C.The activated substrates were placed in a solution containing 20micrograms/ml of a rabbit polyclonal antibody to Streptococcus Group Ain 0.1M HEPES buffer, pH 6.0. Wafers were submerged in the antibodysolution for 60 minutes at 25° C., removed, rinsed with deionized water,and dried under a stream of nitrogen. A thickness or optical densitychange is directly observed by examining a reacted wafer with the SagaxComparison Ellipsometer.

Table 11 summarizes the results obtained with variously coatedfilm-forming particles. Table 11 indicates the antibody concentration,particle concentration, and the addition of any blocking materials ifnecessary. The antibody used for coating of the film-forming particleswas the same as that used as the receptive material on the ellipsometricwafer. Antibody was coated to the surface of the particle by incubationof the mixtures at 25° C. for 16 hours. TC7 preparations were examinedas dilutions of the stock solutions, dialyzed against water and treatedwith a mixed bed resin prior to reaction with antibody.

                  TABLE 11    ______________________________________                 Antibody            %    Concen-  A:S*           Values    Amplifier Solid  tration  Ratio                                   Blocker Neg  Pos    ______________________________________    TC7       3      150      1:1  --      45   60    TC7 (dialyzed)              3      115      1:1  --      45   45    TC7       3      7.7      1:1  --      98   110    TC7 (Ion  3      115      1:1  --      43   50    Exchange)    TC7       3      150      1:1  0.005% SDS                                           63   60    SA:COOH   3      150      1:1  --      145  181    SA:COOH   3      150      1:1  --      143  180    SA:COOH   1      150      1:1  --      74   80    SA:COOH   3      150      1:1  Casein  50   50                                   1.5 15 ug/ml    SA:COOH   3      7.7      1:1  --      132  150    SA:COOH   1      7.7      1:1  --      87   97    SA:COOH   3      150      2:1  --      122  147    SA:COOH   3      150      1:4  --      42   51    SA:COOH   2      150      1:1  --      47   58    SA:COOH   2      150      1:4  --      55   61    SA:COOH   2      150      1:2  --      34   68    SA:COOH/H.sub.2 O              7      150      1:1  --      56   60    SA:COOH/H.sub.2 O              7      200      1:1  --      56   63    SA:COOH/EDC/              7      150      1:1  --      38   42    H.sub.2 O    SA:COOH/EDC/              7      200      1:4  --      48   51    H.sub.2 O    SA:COOH-  1      150      1:1  --      122  82    Surfactant    SA:COOH-  1      150       1:10                                   --      85   90    Surfactant    SA:COOH-  20     370      1:1  --      283  150    Surfactant    SA:COOH-  20     370      1:4  --      280  231    Surfactant    SA:COOH-  20     370      1:1  1:1     213  133    Surfactant                     1% BSA    SA:COOH-  5      150      1:1  --      117  91    Surfactant    SA:N═N/Glut              4      150      1:1  --      55   101    H.sub.2 O    SA:N═NH              10     150      1:4  --      281  135    SA:N═NH              10     150       1:10                                   --      242  111    SA:N═NH              3      150      1:1  --      19   25    Bicine    SA:N═NH H.sub.2 O              4      150      1:2  --      41   63    SA:N═NH H.sub.2 O              10     150      1:1  --      125  217    SA:CONH.sub.2              3      150      1:4  --      66   55    SA:CONH.sub.2              10     150      1:4  --      66   63    SA:N═NH.sup.a              3      300      1:2  --      0    613    SA:CONH.sub.2.sup.a              3      300      1:2  --      0    320    ______________________________________     *Amplifier to Sample Ratio.     .sup.a The final concentration of hydrazide in this particle preparation     was 3M and antibody was coated to the particle in 50 mM MES, pH = 6.0 and     at 56° C. for 30 minutes.

Covalent attachment of the antibody was examined with the carboxylateparticles by the addition of a 1% final concentration (w/v) of acarbodiimide (1-cyclohexyl-3-(2-morpholino-ethylcarbodiimidemetho-p-toluenesulfonate; Aldrich Chemical, Co., Milwaukee, Wis.,Catalog Number C10,640-2, Lot Number 09915PW). The carbodiimide wasadded prior to the addition of the antibody. Hydrazide treated amideparticles-may be treated with a final concentration of 0.05%glutaraldehyde immediately before the addition of antibody to providecovalent attachment of the antibody onto the surface of the particle.Particles were coated with antibody in a 0.01M phosphate buffered salineat pH 7.2 unless otherwise denoted.

The Strep A antigen preparation used for the production of the positivecontrol was commercially available. This antigen was diluted into amixture of one part 2M NaNO₂, one part 2M Acetic Acid, and one part0.66N NaOH to a 1:600 dilution level. The same preparation minus theantigen was used as the negative control. The amplifying reagent wasmixed in varying ratios, as designated in Table 11, with the positiveand negative controls and five microliters were applied to the surfaceof the antibody coated wafer. The samples were incubated for 2 minutesat 25° C., rinsed, and then dried under a stream of nitrogen. Reactedwafers were examined with the modified Sagax Ellipsometer and thethickness was recorded as a change in intensity, measured in millivolts.

These data demonstrate that an extremely high particle density in theamplifying reagent introduces a non-specific association of that reagentwith the test surface. Addition of ancillary proteins or surfactant donot improve the performance of the antibody coated particles. The TC-7particles being slightly more rigid than the Surface Activators do notperform as well for this particular application, however, in comparisonwith some other latex preparations the TC-7 particles demonstratesignificant reactivity. Increased temperature appears to improve thelevel of antibody incorporation into the particle and thus reduces thelevel of free antibody. While free antibody was not removed from theseparticles, it may be advantageous to remove unassociated antibody by atechnique such as ultra-filtration. The hydrazide derivatized amideparticles appear to provide the best overall reactivity although theamide particles also perform well. The surface activator carboxylateparticles do not perform as well as the amide particles.

EXAMPLE 15

Latex

In this study the amplifying film-forming particles were prepared as inExample 14, and the assay was conducted as described there. Thesubstrate employed was a monocrystalline silicon wafer which was lappedwith 12-20 micron aluminum oxide particles, mean particle size 15microns, to create a rough textured surface using a process well knownto those skilled in the semi-conductor industry. This substrate wascoated with a thin film of silicon nitride. A silicon nitride film of350 to 550 Å is standard for this application, however, any filmthickness can be utilized. The T-polymer treatment of the optical slidewas as described in Examples 5 and 14.

The results are shown in Table 12. The visual results differ from thoseobservations made in the ellipsometric system. A very high particledensity provides a clean negative result, but does not produce a strongpositive visual signal. A higher level of antibody incorporated into theparticle produces a stronger signal than does a lower level of antibody,as observed with the instrumented assay. This study suggests thatinsufficient antibody coverage of the particle allows non-specificassociation of the latex particle with the test surface. Once optimized,however, the positive and negative results are readily detectable anddistinguishable.

                  TABLE 12    ______________________________________                 Antibody A:S*   Visual Response    Amplifier  % Solid Concentration                                  Ratio                                       Neg   Pos    ______________________________________    SA:N═NHH.sub.2 O               10      150        1:1  -     +    SA:N═NH H.sub.2 O               10      150        1:1  -     +    SA:N═NH H.sub.2 O               10      150        1:2  -     +    SA:N═NH H.sub.2 O               10      150        1:3  -     +    SA:N═NH H.sub.2 O               10      150        1:4  -     ++    SA:N═NH H.sub.2 O               10      150        1:5  -     +/-    SA:N═NH H.sub.2 O                3      150        1:1  +/-   ++    SA:N═NH H.sub.2 O                3      150        1:5  +/-   +    SA:N═NH.sup.a                3      300        1:2  -     +++    SA:CONH.sub.2.sup.a                3      300        1:2  -     ++    ______________________________________     *Amplifier to Sample Ratio.     .sup.a Final hydrazide concentration is 3M and antibody is added to the     particles in 50 mM MES, pH = 6.0 at 56° C. for 30 minutes. A 2.5M     MOPS was used as the neutralizer in these experiments to provide a final     pH of 8.0.

EXAMPLE 16

Enzyme Amplification

Horseradish peroxidase (Sigma grade VI) was chemically coupled toimmunoglobulins purified by caprylic acid precipitation from pooled hightiter sera from rabbits previously injected with suspensions of cellsfrom cultures of Neisseria meningitidis A, C, Y, W₁₃₅. The coupling wasdone using the reagent S-acetyl thioacetic acid N-hydroxysuccinimideester and methods described in Analytical Biochemistry 132 (1983) 68-73.The resultant conjugate contained peroxidase (104 μM) and immunoglobulin(35 μM) in a buffer of MOPS, 50 mM, pH 7.0. Theperoxidase-immunoglobulin conjugate was diluted in MOPS buffer togetherwith casein (5 mg/ml) and mixed with an equal volume of a dilution of acell-free filtrate from a culture of Neisseria meningitidis organisms.

The mixture (25 μl) was pipetted to the surface of a silicon wafercoated with layers of silicon nitride, t-polymer siloxane, and purifiedimmunoglobulin from the same rabbit antibody preparation to Neisseriameningitidis. Antibody was coated to the T-polymer/silicon wafer from asolution containing 10 μg/ml of antibody in 50 mM MOPS, pH 7.0. Thewafer remained in the antibody for 1 hour at ambient temperature, wasrinsed with deionized water, and dried under a stream of nitrogen. Theantibody coated substrate was further treated by incubating the coatedsubstrate in 0.5 mg/ml hydrolyzed casein in 50 mM MOPS pH=7.0 for 1 hourat ambient temperature followed by rinsing and drying.

Sample was mixed 1 part with 1 part of conjugate. Ten microliters wasapplied to the test surface. After 2 minutes the sample was washed offwith water and the wafer was dried with a stream of nitrogen or blottedwith a filter device. TMBlue precipitating substrate (TMBlue is acommercially available product, trademarked by Transgenic Sciences, Inc.and disclosed in U.S. Pat. No. 5,013,646) was applied to the same areaof the wafer and allowed to stand for 5 minutes. The wafer was washedand dried. A purple spot was visible where the reaction had occurred.This resulting precipitate was then read by eye and ellipsometer toconfirm the presence of N. meningitidis. A 1:20,000 dilution of theantigen is clearly resolved from the negative by eye (see, Table 13).

                  TABLE 13    ______________________________________    N. Meningitidis Results                             Ellipsometric    Fold Dilution* Visual Score                             mVolts    ______________________________________    0              -         64.2     1:10,000      +         152.0    1:5,000        +++       238.5    1:2,500        +++       395.7    1:1,000        ++++      635.0    ______________________________________     *Dilution of the stock antigen preparation into 50 mM MOPS.

A test kit can be formed based on the above assay. This kit contains allthe components necessary to perform up to 50 optical immunoassay rapidtests. The kit features a solid support test station which is designedto facilitate the proper washing and drying steps required. A slide,which may include from one to five (or more) unreacted test surfacesspecific for the conjugated analyte of interest, is placed on the teststation. Upon completion of the first reaction, the slide is tiltedforward away from the operator. The test surface(s) is vigorously rinsedwith wash solution which drains from the tilted surface into thereservoir below. (The reservoir contains a solid absorbent block ofcellulose acetate treated with a biocide.) The slide is then returned toa level position, and a piece of absorbent paper is placed directly ontothe test surface. Several seconds contact time is allowed for fullwicking. The absorbent papers are provided as pads of individualtear-off sheets conveniently located on the front of the test kit, butthe wash/dry process can be effected by alternate means, such ascapillary action. In addition, a solution of an enzyme-labeledsubstance, an enzyme-labeled antibody which is specific to an analyte ofinterest (such as an antigen), is provided, suitably buffered anddiluted. Finally, precipitating means, such as a container ofcommercially available TMBlue liquid, is provided in a convenient volumeso that one to three drops or more can be applied dropwise to cause theenzymatically produced mass change to precipitate before washing. Thesecond incubation is started by adding substrate to the surface and thewash/dry process is repeated to complete the test.

Two different types of silicon wafers were used; one a gold coloredsilicon nitride-coated wafer and the other was a silver-colored siliconwafer without a nitride coating. One possibility is that the visualcolor which is observed with the peroxidase/precipitating substratesystem on the silicon nitride is strictly due to the absorbance of thedye precipitated on the surface. If this is the case and theprecipitated dye is not behaving as a thin film, then the silver-coloredsilicon wafer will produce a visual signal which is the deep blue of theTMBlue only.

T-polymer coated wafers were treated with antibodies for five separatetests; N. meningitidis A, C, Y, W₁₃₅ ; N. meningitidis B; StreptococcusB; H. influenza B; and Streptococcus pneumoniae. The first reagentproduced for each test was wafers, gold and silver, coated with theattachment layer (T-polymeric siloxane) as previously described. Allfive antibodies were coated to these types of wafers at a 10 μ/mlconcentration of antibody in 50 mM MOPS, pH 7.0 by immersing wafers inthe appropriate solution for one hour at ambient temperature. Waferswere rinsed, dried, and blocked as described in Example 1.

The second reagent required utilized the same five antibody preparationsfor the production of antibody-horseradish peroxidase conjugates usingthe method described above. The stock conjugate preparations are used toproduce working conjugate by dilution in 50 mM MOPS, pH 7.0, containing5 mg/ml casein, to a final conjugate ratio of 1:100. One part of theworking conjugate solution is mixed with one part of a standard antigenpreparation, and a 20 μl sample applied to the appropriate antibodycoated wafers.

A rapid protocol was employed using a 2 minute incubation followed by awash, dry then a 5 minute substrate incubation to permit the build-up ofproduct on the wafer surface. Following a wash and blot dry, the samplewas read with both the naked eye and an ellipsometer. Purple coloredspots, strikingly visible, developed on the gold, silicon nitride-coatedwafer, and grey spots were seen using the silver-colored silicon waferwithout nitride coating. On the silicon nitride coated surface a verystrong positive produced a white interference color. The thicknessincrease could be readily measured using the ellipsometer. The visiblecolor produced, in all cases, on the silver wafers, indicate that theprecipitated product behaves as a true thin film and produces aninterference effect even in the absence of an AR coating. The colordeveloped is not dependent on the dye's absorbance characteristics.

Further evidence that the chromogen does not contribute to thegeneration of the observed visual response was gained with the followingexperiment. Treatment of the TMB/H₂ O₂ product with a stopping reagent,H₂ SO₄, produces a yellow precipitate. If the visual response observedwith the optical supports under investigation here is solely due to thechromogen, then the treatment of the surface precipitate with stoppingreagent should yield a yellow-colored spot. Treatment of the immobilizedsurface precipitate with sulfuric acid does not modify the strong purpleor blue spot produced on the silicon nitride coated wafer. Therefore,the resultant signal is entirely dependent on the formation of a thinfilm. Additional verification was obtained by using a strip of adhesiveto remove the precipitate from the surface of the silicon nitride. Theprecipitate removed with the adhesive was a pale grey/blue with no redcomponent. The observed interference effect exhibits a brightpurple/blue color with a strong red component. This re-enforces the ideathat thin film formation is responsible for the generation of theobserved color effect.

Table 14 represents the results obtained from a comparison of the massenhanced assay to the latex agglutination assays manufactured byWellcome Diagnostics for a whole range of bacterial antigen assays.

                  TABLE 14    ______________________________________    Sensitivity Comparison Of OIA with Latex Agglutination                            Latex 1 +      OIA/    Organism  Source of Antigen                            Reaction.sup.a                                     OIA.sup.b                                           Latex*    ______________________________________    N. meningitidis              Cell supernate                              4K       20K 5    A,C,Y,W.sub.135    N. meningitidis B              Kit positive diluted in                             8       32    4              Cerebral Spinal Fluid              Kit positive diluted in                            20       160   8              buffer              Cell supernate                              25K     200K 8    H. influenza B              Kit positive  10       80    8    Streptococcus B              Kit positive  10       50    5              Pronase extract of                              10K      40K 4              cell suspension    S. pneumoniae              Kit positive  100      Neg.  --              Type 4 polysaccharide                            200      400   2              Type 9 polysaccharide                            50       50    1              Type 12 poly- 50       10    0.2              saccharide    Streptococcus A              Positive Antigen                            80       1600  20    ______________________________________     .sup.a Latex Agglutination Assay; commercialiy available.     .sup.b Mass Enhance Catalytic     *Represents relative increase in sensitivity achieved with OIA compared t     latex agglutination.     NOTES:     1) For OIA, the dilution is the last dilution giving a visibly positive     result.     2) Cell supernates noted here are the supernatants removed after overnigh     +4° C. standing of a heavy cell suspension made in 0.5% formalin i     saline. They have a high content of the polysaccharide, hence require     considerable dilution.     3) Latex tests were done with commercially available products which had     not expired.

The results shown in Table 15a and 15b are a comparison of the catalyticmass enhanced method and an Enzyme-Linked Immunoadsorbant Assay (ELISA).The data demonstrate an enhanced performance of the enzyme amplifiedassay for Meningitidis A, C, Y, W₁₃₅ relative to the ELISA, using TMB asa substrate. Production of the ELISA test surface and the optical testsurface are described below.

There are two major differences between these techniques. First, themethod of this invention utilizes a polished silicon wafer for solidphase adsorption of the antibody while ELISA utilizes a clearpolystyrene microtiter plate. Second, and more important, the substratesused to develop the reaction for this catalytic method produce aninsoluble product that deposits on the surface of the polished siliconwafer, while the substrate for ELISA produces a colored solution in thewells of the microtiter plate. It is because of this importantdifference that the results obtained with this catalytic method are moresensitive. The ELISA depends on a visible color to be produced from thechromogen, while the device of this invention depends only on a thinlayer of chromogen to be deposited on the device.

Specifically, one surface is a polished silicon wafer (OIA) and theother surface, a clear polystyrene, microtiter plate (ELISA). Bothsurfaces received a 10 μg/ml antibody solution for 1 hour at roomtemperature, a deionized water rinse, and a 0.5 mg/ml casein blockingsolution for 10 minutes at room temperature, and a final deionized waterrinse.

In the assay, antigen dilutions where:

1:5,000; 1:10,000; 1:20,000; 1:40,000; 1:80,000; and 1:160,000; and theconjugate solution was a 1:100 dilution of HRP labeled antibodycontaining 5 mg/ml casein and 50 mM MOPSO, pH 7.0. One part of eachantigen dilution was combined with one part conjugate solutionimmediately before use and applied to each surface. This was allowed toreact for 2 minutes at room temperature, then each surface was rinsedwith deionized water. Substrate was then added to each surface. Thesilicon wafer received TMBlue and the ELISA plate received TMB. This wasallowed to react for 5 minutes at room temperature. At this point, thereaction was over and the silicon wafer was rinsed with deionized waterand dried with nitrogen. The ELISA was stopped with H₂ SO₄. A visualreading was made to determine the lowest antigen dilution differentiablefrom the negative and the test surface containing the insoluble productdeposited on the surface put into the ellipsometer to measure therespective voltages.

OIA could be read out to a 1:40,000 antigen dilution as compared toELISA (unstopped) which could be read to only a 1:10,000-1:20,000dilution, while ELISA (stopped) could only be read to a 1:5,000-1:10,000dilution by eye.

Instrument read results are shown in Tables 15a and 15b.

                  TABLE 15a    ______________________________________    Catalytic Mass Enhanced Method (OIA) ™ Results                              Change in    OBS.sup.A   Fold Antigen Dilution                              Intensity*    ______________________________________    2            0.000        0.006    2            5,000        0.339    2           10,000        0.154    2           20,000        0.059    2           40,000        0.023    2           80,000        0.013    2           160,000       0.011    ______________________________________     .sup.A Number of observations made.     *Change in intensity is actual intensity minus background intensity     recorded with the Comparison Ellipsometer.

                  TABLE 15b    ______________________________________    ELISA Results             Optical Density Readings at 450 nM    Dilution   1       2          3     4    ______________________________________    0          0       0.013      0.003 --    1:160,000  0       0          0.008 0.015    1:80,000   0.006   0.036      0.037 0.045    1:40,000   0.006   0.005      0.027 0.001    1:20,000   0.015   0.012      0.012 0.041    1:10,000   0.030   0.043      0.068 0.053    1:5,000    0.081   0.085      0.097 0.123    1:5,000    0.063   0.064      0.094 0.088    ______________________________________

EXAMPLE 17

Latex and Catalytically Enhanced Assays

An enzyme-labeled assay was used to detect antigen from Streptococcus Aand compared on the same silicon wafer with an assay using the amidemodified surface activator latex, 0.161 μm (Rhone-Poulenc).

Both techniques are more sensitive than a commercially available(Wellcome Diagnostics) latex agglutination technique which has a cut-offat a 1:80 dilution of antigen. A direct instrumented comparison of thetwo techniques is presented below in Table 16. The mVolt readings givenare a function of a change in light intensity recorded with the modifiedSagax Comparison Ellipsometer.

                  TABLE 16    ______________________________________    Fold Antigen Dilution                   mVolts/Latex                              mVolts/Enzyme    ______________________________________    0              3.0        11.0     1:320         32.0       203.0     1:160         63.0       290.0    1:80           113.0      272.0    1:40           195.0      194.0    1:20           316.0      168.0    1:10           428.0      258.0    ______________________________________

EXAMPLE 18

Multiple Analyte Protocol

Test surfaces and conjugated antibody preparations were produced asdescribed in Example 16 for each of the following organisms, N.meningitidis, H. influenza Group B, Streptococcus pneumoniae,Streptococcus Group B, and E. coli K1. The test device was designed toaccommodate these five test surfaces which were mounted onto theelevated platforms within the device (see, FIGS. 9A-E and 11A-E). Afixed volume of individual conjugate preparation for each of thefollowing organisms, N. meningitidis, H. influenza Group B,Streptococcus pneumoniae, Streptococcus Group B, and E. coil K1 wasprepared.

Equal volumes (75 μl) of a cerebral spinal fluid (CSF) sample and thisconjugate preparation were mixed before pipetting one drop(approximately 25 μl) onto each of the five antibody coated testsurfaces. The CSF samples were prepared with known levels and knowncombinations of antigens derived from the test organisms, as describedin Example 16. Samples were incubated for 2 minutes, after which thetest wafers were washed with deionized water and blotted dry. Substrate(TMB precipitating reagent) was added to each surface and incubated for5 minutes. The wafers were washed with deionized water, blotted dry, andread. In this manner, a single sample was easily analyzed for thepresence of one or more analytes. This poly-specific reagent maintainedthe specificity observed with the mono-specific reagents. No falsepositive responses were observed, and positive responses were comparableto the signal produced in a mono-specific test procedure.

EXAMPLE 19

Strep A Assay Device

The details of formation of the device shown in FIGS. 8A-8G is nowprovided. Monocrystalline silicon wafers, 100 mm in diameter, polishedon one side, 20 mil±2 mil were purchased from a semi-conductor supplier.The wafers were coated with 495 Å±15 Å silicon nitride or titaniumdioxide using processes described above. Each wafer was sawed to a depthof 3.5 mils generating a pattern of 0.75 cm² sections. This allows thewafers to remain intact for subsequent processing. The wafers were thencoated with the T-polymer siloxane as described in Example 5. A finalpolymer thickness of 100 Å±5 Å is used. Polymer coated wafers are curedfor 24±2 hours at 145° C.±5° C.

Polymer coated wafers were submerged in a solution containing 5 μg/ml ofan affinity purified rabbit anti-Strep A antibody in 0.1M HEPES (N-2-Hydroxyethyl! piperazine-N'- 2-ethanesulfonic acid!) at pH 8.0. Waferswere coated with the antibody solution for 16-20 hours at 2° C.-8° C.The wafers were rinsed with deionized water and then dried under astream of nitrogen. A procedural control was applied to the center ofeach 0.75 cm² using an x,y translational stage. A 1-2 μl spot wasapplied and incubated for 3 minutes at ambient temperature (20° C.). Theconcentration of antigen used was empirically determined to provide anintermediate color change. Antigen was mixed in deionized water. Theantigen solution was rinsed from the surface with deionized water andthen dried under a stream of nitrogen. The wafers were then submerged ina solution containing 0.5% of degraded gelatin in 50 mM MOPS (3-N-Morpholino!propanesulfonic acid), pH 7.0 for 20 minutes at ambienttemperature. The wafers were removed from the solution and dried under astream of nitrogen. The gelatin layer serves to stabilize the antibodycoating, and aids in storage of the device. The wafers are purple incolor. The gelatin layer was fully hydrated by exposure of the wafers tosteam for 30 seconds. The wafers were then air dried. The wafers willreturn to the original gold color. The wafers were then broken intoindividual 0.75 cm² test pieces.

Referring to FIGS. 8A-8G, the molded test device has the pre-cutabsorbent pad placed in the bottom and the protective cover snapped intoplace. The upper laminate of blotting materials was placed in the lid ofthe device and the protective cover snapped into place. A small drop ofepoxy is applied to the raised pedestal in the center of the device anda test surface applied. The glue was allowed to set, and then the deviceis closed and placed in the kit.

The antibody preparation used to coat the surface, or a separateantibody preparation, was conjugated to HRP using standard periodatechemistries described by Nakane. The exact dilution of conjugatedantibody used in the mass enhancement reagent will depend on the levelof HRP incorporation and the affinity and avidity of the antibodypreparation used. The conjugate preparation was diluted in a solutioncontaining 50 mM MOPSO (3- N-Morpholino!-2-hydroxypropanesulfonic acid),pH 7.0, 20 mg/ml of standard alkaline treated casein, 0.3% (v/v) Tween20, and 0.5% (v/v) Proclin 300 (Rohm and Haas). Conjugate solution wasdispensed into Wheaton Natural polyethylene dropper bottles. Delivereddrop size was approximately 30 μl. All other reagents were alsodispensed into the Wheaton Natural dropper bottles.

Extraction tubes were prepared by dispensing 100 μl of a solutioncontaining 2.3M NaNO, and 0.01% isopropanol into polyethylene tubes andallowing the solution to dry onto the tube. This may be accomplished bydrying at ambient temperature, under circulating air, or at 45° C.

The reagent composition was as follows:

Reagent #1: 0.25M Acetic Acid with 0.035 mg/l bromcresol green

Reagent #2: 1.5M MOPSO, pH=7.3, with 0.2% (v/v) Tween 20, 0.5% (v/v)Proclin 300, and 20 mM EDTA.

Reagent #3: Conjugate.

Reagent #4: Deionized water containing 0.1% Proclin 300.

Reagent #5: Commercial preparation of precipitating TMB.

The test procedure for use of this device was as follows.

1. Remove reagent(s) from refrigerated storage and allow to warm to roomtemperature.

2. Remove an extraction tube containing dry reagent from the kit andplace it upright in a rack or holder.

3. Label extraction tubes and test devices with appropriate patientinformation. Place test devices on a level surface while the assay isbeing performed.

4. Add 3 drops of Reagent 1 into the extraction tube and shake it gentlyto dissolve the dry reagent in the bottom. The liquid should becomelight green in color when dissolved and properly mixed. Color change inthe extraction tubes may not be evident with bloody specimens, howeverassay performance is not affected.

5. Within 1 minute, place a throat swab containing a specimen orpositive control into the tube. Press the swab against the sides of thetube while rolling the swab so that the liquid is moved in and out ofthe fiber tip. Allow the swab to incubate in the extraction solution fora minimum of 2 minutes.

6. Hold the swab shaft to the side and add 3 drops of Reagent 2 directlyinto the extraction tube. Use the swab to mix the reagent with theextract until the solution color changes from green to blue.

7. Separate the extraction solution from the swab by rolling the swabagainst the wall of the extraction tube while squeezing the sides of thetube as the swab is withdrawn. Discard the swab and retain the contentsof the tube. Retain as much fluid from the swab as possible.

8. Add 1 drop of Reagent 3 to the extract and mix thoroughly. Do not letstand more than 30 minutes.

9. Use a clean transfer pipette to transfer 1 drop of the solutiondirectly onto the center of the surface of the corresponding testdevice. Do not cover the entire surface of the test device.

10. Incubate the drop on the test surface for 2 to 5 minutes.

11. A vigorous wash of 1-2 second duration is important. Rinse thesurface of the device using Reagent 4 wash solution with care not toexceed the capacity of the absorbent material surrounding the device.

12. Confirm that the blotting device is in position #1. Close the lid ofthe device momentarily to remove residual moisture from the surface.Blot with a clean surface each time blotting is necessary. Blottershould be in position I when blotting for the first time. If in positionII, move to position I for the second blot. Repeated blotting in thesame position may compromise test results.

13. Open the lid and apply 1 drop of Reagent 5 directly onto the centerof the surface of the test device and incubate for 4 to 10 minutes. Ifplacement of the first drop was not directly onto the center of thedevice, place the Reagent 5 drop directly over the area of the firstdrop.

14. Vigorously rinse the surface of the device for 1-2 seconds usingReagent 4 wash solution.

15. Move the blotter in the top of the device to position #2 and closethe lid of the device momentarily to remove residual moisture from thesurface. Open the lid and examine the test surface for a color change.

POSITIVE RESULT

Solid blue/purple colored reaction circle of any intensity appears inthe center of the device surface.

NEGATIVE RESULT

No blue/purple colored reaction circle of any intensity appears on thetest surface.

A Procedure control is present on each test surface. It appears as asmall blue/purple dot in the center of the test surface upon completionof each positive or negative test. A negative test result will show onlythe procedure control. A positive test result will show the procedurecontrol within the reaction circle. With very strong positive results,the procedure control may be less apparent within the reaction circle.

If the procedure control does not appear, the procedure can be repeatedfollowing the instructions. The reacted test surface and the colorchange associated with a positive reaction will not deteriorate overtime. Therefore, the test device may be considered a permanent record.If a test device is to be saved for reference, the blotting material inthe lid should be removed and disposed of in a biohazard container, andthe device should be closed for storage.

EXAMPLE 20

Sensitivity of OIA Device

The analytical sensitivity of Strep A OIA was compared with commerciallyavailable Strep A kits using a cell suspension of a known density andextracting an aliquot of this suspension according to each kit's assayprotocol. Streptococcus pyogenes, Lancefield group A, was obtained fromthe American Type Culture Collection (ATCC #12344) as primary culture ona slant tube. Cell suspensions were made sterile normal saline andserially diluted with the normal saline. Results from the studydemonstrate at least a 10-100 fold greater sensitivity in the OIA deviceof this invention compared to at least six commercially available testkits.

Table 17 compares the clinical sensitivity of the Strep A OIA to theproduct insert claims of several commercially available rapid Strep Aassays. The majority of these rapid assays discard all samples withStrep A colony counts of less than 20, however, in the numbers presentedfor the Strep A OIA assays none of the samples were discounted. Strep AOIA demonstrates a significant improvement in sensitivity relative tothese rapid tests.

                                      TABLE 17    __________________________________________________________________________    Comparison of Test kits for Detection of Group A    Streptococcal Antigen Directly from Throat Swabs.sup.1    Product Name/   Assay Time               Predictive Value    Manufacturer             Methodology                    Extraction/Reaction                              Sensitivity                                   Specificity                                        Accuracy                                             pos  neg    __________________________________________________________________________    STREP A OIA             Mass enhanced                    2-10 min                         6 min                              100%.sup.2                                   98.9%                                        99.1%                                             100% 98.9%    BioStar Medical             on a silicon    Products, Inc.             surface    OIA Strep A             Ab coated latex                    2-10 min                         2 min                               83.3%.sup.2                                   97.4%                                        94.7%                                              88.2%                                                  96.1%    BioStar Medical             on silicon    Products, Inc.             surface    Test Pack +             Ab coated                    1-30 min                         5 min                               93.7%                                   94.5%                                        94.3%                                              89.7%.sup.3                                                  96.7%.sup.3    Strep A  colloid on    Abbott Labs             membrane    Cards -OS Strep A             Ab coated,                    2-60 min                         5 min                               96.6%.sup.4                                   96.8%                                        96.7%                                              95.5%                                                  97.6%    Pacific Biotech,             dyed latex    Inc.     on membrane    Directigen 1, 2, 3             Ab coated,                    3-120 min                         not   90% 92%  91.2%                                              75%.sup.3                                                  97%.sup.3    Strep A  dyed liposome                         given    Becton-Dickinson             on membrane    Reveal Colour             Ab coated,                    2 min                         3 min                               86% 94%  94%   81% 96%    Strep A  dyed latex, w/rocking    Wellcome agglutination    Diagnostics    __________________________________________________________________________     .sup.1 Data obtained from manufacturers' package inserts.     .sup.2 Based on all specimens.     .sup.3 Data calculated from tables in package inserts.     .sup.4 Specimen plate colony count not revealed in package insert.

Swabs were collected from patients presenting symptoms of pharyngitisusing a single swab and standard specimen collection techniques. Fourindependent laboratories were used in the study. Immediately after thespecimen was collected, the swab was returned to the transport tube andthe capsule containing transport media crushed. Each site inoculated a5% Sheep blood agar (SBA) plate with a specimen and plates were thenincubated at 35° C.-37° C. Two of four sites incubated the pates underanaerobic conditions for 24 to 48 hours and two sites incubated theplates in an enriched CO₂ environment. Each site reported results asnegative or positive with positives confirmed by a serotyping method.

After inoculation, the swab was returned to the transport tubes andassayed following the Strap A OIA test procedure. Three drops of acidgenerating solution were added to extraction tubes and mixed well todissolve a dried reagent. The swabs were placed in the extraction tube,thoroughly saturated with extraction reagents, and allowed to incubatefor 2 minutes in the solution. Three drops of neutralizing solution wereadded to the extraction tubes and the swabs were used to mix thereagents. The extracted swabs were expressed against the side of thetube, then discarded. This extraction technique is common to most rapidGAS tests and liberates GAS antigen from the bacterium.

One drop of catalyst was added to the extract and mixed thoroughly. Adrop of this solution was applied to the center of the test piece (FIGS.8-8G). Samples were incubated on the test surface for 2 minutes, thenthe test surface was rinsed with water and blotted dry by closing thelid. A drop of mass enhancer was applied to the center of the testsurface and incubated for 4 minutes. The surface was again rinsed withwater and blotted as before. The test results were determined withoutknowledge of the culture results.

An enhanced culture method was designed to confirm the presence ofbacteria. In this method, the pledgets (plugs separating transport mediaand swab) were removed from all transport tubes, placed in Todd-Hewittbroth and incubated for 24-48 hours at 35° C.-37° C. If growth patternsconsistent with the bacteria were observed, colonies were selected,re-isolated if necessary, and confirmed using a commercially availableStreptococcus serotyping kit.

In a clinical study a total of 778 samples from four sites wereexamined. The SBA culture method determined 70 specimens to be positive,4 of these specimens were determined to be negative relative to theenhanced culture method. The enhanced culture method determined that 92specimens were positive. The sensitivity of SBA culture was 71.7%relative to the enhanced culture method for the frequency and populationtested. These data support previous literature results showingconventional SBA culturing methods to be less sensitive than enhancedculture methods. Therefore, the consideration of conventional SBAculture methods as the "gold standard" should be re-evaluated.

OIA results were evaluated relative to both SBA culture and enhancedculture methods. The Strep A OIA yielded a sensitivity of 92.9%, aspecificity of 94.8%, and an accuracy of 94.6% relative to the SBAculture, for the frequency and population tested. Strep A OIA appears tolack specificity relative to the SBA culture methods. However, theactual limitation lies in the SBA culture technique as 26 of theapparent Strep A OIA false positives were, in fact, true positives. Thesensitivity of the Strep A OIA relative to culture appears to be reducedbecause of 4 SBA positive results which were Strep A OIA negative. Theseresults were later determined to be culture non-isolates by the enhancedculture method. Strep A OIA detected 91 out of 92 enhanced culturepositives yielding a sensitivity of 98.9%. It is important to note thatthe performance results include all data collected, irrespective ofcolony count.

EXAMPLE 21

Instrument Reading Protocols

1) Photodiode Modified Comparison Ellipsometer

The Comparison ellipsometer was modified as previously described above.The eyepiece was connected to a CCD camera to allow samples to becentered in the elliptical reticle. The zoom was adjusted so that thesample spot is completely enclosed in the ellipse.

The test strip used in the assay was 1 cm wide and 4-5 cm in length.These dimensions are easy to handle manually. Any dimension of testpiece may be used with the proper design of sample positioning devices.Samples were applied as 20 μl drops evenly spaced along the length ofthe slide. One section was left for measurement of the test surfacebackground. Samples were assayed as described in the previous examples.

The test strip was placed on the instrument's sample platform and thebackground section of the surface was centered in the ellipse. Thesample platform has x,y positioning capabilities. Once the test surfacewas positioned, the background reading was taken at the photodiode. AnLED displays the background intensity of the background section involts. Computer software may also be designed to record the backgroundintensity. After this measurement was complete, the platform wasadvanced to record a reading from a negative sample. The voltage wasrecorded directly, or may be recorded as sample minus background. Theplatform was advanced until all samples were measured.

The instrumented read out may provide a qualitative answer of yes or norelative to a pre-set signal. The assay would include a negative controland a low positive, or cut-off concentration and objectively evaluatesamples relative to this threshold value.

If the assay is quantitative, the test surface or test device will allowmeasurement of a negative control and one or more known positivecontrols. Samples values will be compared to this curve forquantitation. Positive controls may cover several broad ranges if asemi-quantitative answer is adequate for the application beingconsidered.

Monochromatic Light Source, Comparison Ellipsometer

This instrument has a smaller optical path due to the use of a lightsource that is collimated, a smaller reference surface, a smaller sampleplatform, polarizers positioned immediately next to the light source andthe detector, and eliminating the lens system required for visualexamination of the surface.

The reading protocol for this particular instrument accommodates fiveseparate samples or four controls and one sample or two controls andthree samples, etc. The sample slide is connected to a rotating postwhich controls the slides position. Alignment is not achieved by visualplacement, but by sample placement on the test surface and the platformadvancement. Any arrangement of the test surface may be used bymodifying the x,y positioning platform. This allows the use of a testsurface which can examine a large number of samples.

This instrument uses a photodiode detector which is masked to match afixed sample size and the slide positioning and sample applicationallows the sample spot to fill the mask. Readings are made from an LEDdisplay in the cover of the instrument. Readings are in millivolts.

EXAMPLE 22

Group B Streptococcus

An optically active test surface was produced by immobilizing either apolyclonal or monoclonal anti-GBS antibody on the previously describedsiloxane coated surface. The antibodies were group specific, but may beto any of the group specific epitopes found on the GBS polysaccharide.The polyclonal preparations used were a protein G purified IgG fractionthat was adsorbed to whole cells. The coating antibody concentration wasin the range of 150 to 500 μg of antibody per 100 mm wafer, and wascoated at 2-8 degrees C. for 12 to 16 hours. The 100 mm wafer may or maynot possess a 495 Å silicon nitride. In particular, a monoclonalantibody was found to coat best at 200-300 μg/wafer in a buffercontaining 50 mM Sodium Acetate at pH 5.0. A polyclonal antibody wasfound to coat the surface equally well but in a buffer containing 0.1MHEPES, pH 8.0. Buffers covering the pH range of 5.0 to 9.0 have beendemonstrated to provide an effective antibody coating. Once the antibodywas immobilized on the test surface, an overcoat of 0.5% degradedgelatin in 50 Mm MOPS, Ph 7.0 is applied as previously described.Antibody coated test surfaces were placed in single test devices priorto use.

For mass enhancement of the GBS assay, a group specific polyclonalantibody preparation was conjugated to HRP using the Nakane periodatemethod. Conjugation of the antibody to HRP at high Ph, greater than9.75, resulted in an increase in non-specific binding. Optimalconjugating Ph was established at 9.0 to 9.75.

The GBS group specific antigen was extracted from the organism using anacid extraction protocol. A mixture of 0.25M acetic acid and 2.3M sodiumnitrite was used to generate hyponitrous acid. The acetic acid was foundto effectively extract the GBS antigen in the range of 0.1M to 1.0M.Antigen was extracted from the organism for 2 minutes. For all resultsdescribed here, ATCC strain number 12386 was used. The solution wasneutralized using an buffer containing 1.5M MOPSO, Ph 7.3, 0.2% Tween20,0.5% Proclin300, and 20 Mm EGTA by adding an equal volume of theneutralizing buffer. A final Ph range of 6.0 to 7.5 is desired.

The extracted antigen was mixed 5:1 (sample:conjugate) with a 1:150dilution of the conjugate preparation containing 50 Mm MOPSO, Ph 7.0, 20mg/ml alkaline treated casein, 0.3% Tween20, and 0.5% Proclin300. Asample:conjugate ratio of between 3:1 and 10:1 was acceptable. Thesample/conjugate mixture was incubated for 5 minutes at roomtemperature, then 20 μl of the mixture applied to the test device andincubated for 5 minutes at room temperature. The devices were used aspreviously described for washing and drying the test surface. After thetest surface was dried, a drop of TMB precipitating substrate wasapplied to the device and incubated for 5 minutes at room temperature.After the wash/dry protocol was completed, the test result wasinterpreted. When instrumented results are desired, the wash dryprotocol is accomplished by rinsing the test surface under a stream ofdeionized water and drying under a stream of nitrogen. The initialincubation of sample/conjugate was not required but was observed toincrease the sensitivity of the assay. Assay sensitivity could befurther increased with additional incubation time.

The current GBS assay demonstrated excellent tolerance to changes in thefinal extraction Ph. The visualization of a low positive was noteffected by a change in Ph over the range 6.75 to 8.0, see Table 19. Avisual scale was established for scoring results, wherein a 1+ or 2+ wasa very weak purple spot on a gold background. A value of 10+ is a verystrong blue result.

                  TABLE 18    ______________________________________    GBS Sensitivity as a Function of Assay Time    Sensitivity.sup.a                 Total Assay Time.sup.b    ______________________________________    3 × 10.sup.6                  5.sup.c    3 × 10.sup.4                 10.sup.d    3 × 10.sup.3                 15.sup.e    ______________________________________     .sup.a Sensitivity is expressed in the number of cells/swab.     .sup.b Time is in minutes.     .sup.c Incubation times were 1 minute for sample/conjugate; 2 minutes for     the sample on the surface; and 2 minutes for substrate.     .sup.d Incubation times were 5 minutes each for the sample on the test     surface and substrate.     .sup.e Incubation times were 5 minutes each for the sample/conjugate,     sample on the surface, and substrate.

                  TABLE 19    ______________________________________    GBS DETECTION AS A FUNCTION OF THE FINAL ASSAY pH    CELLS  pH 6.75 pH 7.0  pH 7.25                                  pH 7.5                                        pH 7.75                                              pH 8.0    ______________________________________    3 × 10.sup.6           10+     10+     10+    10+   10+   10+    3 × 10.sup.5           7+      7+      7+     7+    7+    7+    3 × 10.sup.4           2+      2+      2+     2+    2+    2+    0      --      --      --     --    --    --    ______________________________________

EXAMPLE 23

Chlamydia Detection

An optically active test surface, with or without the 495 Å siliconnitride coating, was coated with the polymeric siloxane, T-polymer, aspreviously described. These polymeric coated supports were furthercoated with a solution of between 1 and 10 μg/ml of bovine serum albumin(BSA) in 100 Mm sodium carbonate, Ph 9.6, at 2-8 degrees C. for 12 to 16hours. Subsequently, the test surface was coated with 0.5% degradedgelatin in 50 Mm MOPS, Ph 7.0, at room temperature for 20 minutes andthen dried under a stream of nitrogen. Coated test surfaces were mountedinto the single use test devices previously described.

The Chlamydia specific LPS antigen was extracted from the syntheticfiber swab used for sample collection by immersing the swab in asolution (approximately 120 μl) of 0.01M PBS with 0.1% (w/v)chenodeoxycholic acid (CDOC), sodium salt, which had been alkalized with10 μl in 1 ml of buffer of 1.0N NaOH to a final Ph of 11.5. Initiallythe CDOC was solubilized in absolute methanol at 0.2 g/ml. The swab wasideally vortexed in this solution for approximately 10 seconds and thenincubated for 5 minutes at room temperature. The swab was then expressedof residual solution by squeezing the flexible extraction tube(polypropylene). An equal volume of 100 Mm Na₂ HPO₄ with 0.1% CDOC at Ph7.0 was added to adjust the final pH to between 7.0 to 7.5.

A mass enhancement reagent was prepared by conjugating ananti-Chlamydial LPS antibody to HRP using the method of Nakane. Thestock conjugate was diluted 1:75 to 1:200 in a diluent containing 50 mMMOPSO, pH 7.0, with 20 mg/ml of alkaline treated casein, 0.3% tween20,and 0.5% PROCLIN300®.

Approximately 30 μl of the extracted sample was put onto theBSA/polymeric siloxane test surface and incubated from 5 to 10 minutesat room temperature. Then approximately 30 μl of an anti-Chlamydial LPSantibody conjugated to HRP was added to the sample spot on the testsurface and incubated for 1 to 5 minutes at room temperature. The testsurface was washed with 1 to 2 ml of water and dried in the device aspreviously described. Approximately 30 μl of the TMB precipitatingsubstrate was applied to the surface and incubated for 10 to 15 minutesat room temperature. The test device was washed and dried as previouslydescribed.

Alternatively, an antigen preparation was serially diluted from 1:100 to1:320 in 0.01M PBS, pH 7.4. Three fold serial dilutions were performed.Five μl of these dilutions were added to 500 μl of 0.1% chenodeoxycholicacid in 0.01M PBS with 10 mM NaOH (final pH was 11.5). These werevortexed and allowed to sit for 5 minutes at room temperature. Fivehundred μl of 100 mM phosphate buffer (mono and di basic mix) was addedto neutralize the extraction buffer and 15 μl of this added to the wafersurface. This was incubated on the surface for 10 minutes at roomtemperature. A 15 μl aliquot of a 1:75 dilution of an anti-LPSantibody-HRP conjugate was added directly to the antigen spot on thesurface and incubated at room temperature for the designated time. Thewafer was washed with deionized water and blown dry under a stream ofnitrogen. Substrate (30 μl) was applied to the antigen/conjugate spotand incubated for the designated time. The wafer was washed and dried asbefore and the intensity of captured antigen recorded with thephotodiode modified Comparison Ellipsometer. The data was corrected forbackground. Readings are in millivolts are the average value for 2readings, see Table 20. Table 20 demonstrates the effect of increasedincubation times on the sensitivity of the assay.

                  TABLE 20    ______________________________________    Incubation Times (minutes)            CON-     SUB-     ANTIGEN    SURFACE JUGATE   STRATE   DILUTION                                      mV    VISUAL    ______________________________________    5       5        10       0       12.4  -                               1:320  52.5  1+                               1:160  86.0  1+                              1:40    109.5 3+                              1:20    708.0 5+    10      5        15       0       -6.3  -                               1:320  181.5 2+                               1:160  172.9 2+                              1:40    861.1 5+                              1:20    1601.4                                            10+    ______________________________________

EXAMPLE 24

Human Anti-HIV Detection

A silicon wafer, 100 mm diameter, was coated with polybutadiene (PBD) aspreviously described. The PBD coated optical test surface was coatedwith 20 μg/ml of a synthetic peptide corresponding to GP41 of the HIVvirus which was conjugated to bovine serum albumin (BSA). This antigenpreparation was diluted in 10 mM Potassium Phosphate, 0.85% NaCl, at pH7.0, and the test surface was coated with the antigen for 2 to 3 hoursat room temperature with agitation. The test surface was removed fromthe coating solution, rinsed with deionized water, and dried under astream of nitrogen. The gp41 protein is very hydrophobic and binds wellto the hydrophobic PBD surface.

A series of seronegative and seropositive human serum samples were usedto test the GP41/BSA coated surface. Five microliter samples of serumwere applied to the test surface and incubated for 15 minutes at roomtemperature. Antibody capture on the antigen surface was measured usingthe Comparison ellipsometer modified with a CCD camera. Results werereported in Grayscale units.

                  TABLE 21    ______________________________________    Host Antibody Capture on a gp41 Surface    Seropositive Serum Grayscale                     Seronegative Serum Grayscale    ______________________________________    93.07            8.83    43.53            3.65    48.64            5.51    60.29            7.64    65.36            3.62    ______________________________________

The average value reported for a seronegative serum was 5.85±2.1grayscale units making any value of 12.15 grayscale units(negative+3standard deviations) a positive response. The varying level of grayscalereported for the seropositive samples reflects varying antibody titers.

A recombinant antigen preparation which was a fusion of gp41 and p24 ofthe HIV virus was also evaluated as a coating material for the opticaltest surfaces of this invention. A 100 mm diameter silicon wafer wascoated with PEI as previously described. This optical test surface wassubmerged in a coating solution containing 2.5 μg/ml of the fusionprotein in 50 mM sodium carbonate, pH 8.0 and incubated for 3 hours atroom temperature. The optical test surface was then rinsed withdeionized water and dried under a stream of nitrogen. The optical testsurface was tested for response to serum controls: negative, low,medium, and a high positive anti-HIV. Five microliters of each serumsample was applied to the surface and incubated for 15 minutes at roomtemperature. Surfaces were rinsed with deionized water and dried under astream of nitrogen. Results were obtained with the modified ComparisonEllipsometer as described above.

                  TABLE 22    ______________________________________    Antibody Capture by gp41/p24    Serum Sample  Grayscale Value    ______________________________________    Negative      0.0    Low           29.28    Medium        41.22    High          52.94    ______________________________________

EXAMPLE 25

RSV Detection

Optically active test surfaces were coated with the polymeric siloxaneas previously described. These surfaces were placed in a 10 μg/mlpolyclonal anti-RSV antibody solution. A number of buffers were examinedin the antibody coating step, including HEPES, pH 8.0; Acetate, pH 5.0;Carbonate, pH 9.5; Borate, pH 8.0; and Phosphate, pH 7.4. All bufferswere 0.1M in concentration. The 0.1M sodium carbonate, pH 9.5 was foundto be optimal. Surface preparations were analyzed using a sequentialassay. A 20 μl aliquot of positive control was applied to the surfaceand incubated for 10 minutes at room temperature. Surfaces were rinsedwith deionized water and dried under a stream of nitrogen. Antibody-HRPconjugate (20 μl) was applied to the test surface, incubated 10 minutes,washed, dried, and then 20 ρl of precipitating substrate applied for 10minutes, followed by a wash/dry step. Antibody was coated for 12 to 16hours at 2-8 degree C. The surface was rinsed and incubated for 20minutes at room temperature in a solution containing 0.5% degradedgelatin, 50 mM MOPS, pH 7.0 and then dried under a stream of nitrogen.

The mass enhancement reagent was prepared by the Nakane method. Amonoclonal antibody to the nuclear peptide of RSV was conjugated to HRP.The stock conjugate was diluted 1:30 to 1:250 in a solution containing50 mM MOPSO, pH 7.0, and 20 mg/ml alkaline treated casein, 0.3% Tween20,and 0.5% Proclin300.

An alternate assay protocol involved thoroughly mixing 30 μl of a nasalwashing with 30 μl of 10% Tween20 in PBS and 30 ml of a 1:40 dilution ofthe conjugate. A 30 μl aliquot of this sample was applied to the testsurface and incubated for 12 minutes at room temperature. The rinse anddrying protocols-described for the single use test device were used. TMBprecipitating substrate was applied to the surface, incubated for 8minutes at room temperature, rinsed, and dried prior to interpretation.

                  TABLE 23    ______________________________________    RSV Standard Curve    Fold Dilution                Ellipsometric Intensity (mV)                                 Visual    ______________________________________    0           0.0020           -     1:1280     0.0244           -     1:640      0.0256           +     1:320      0.0356           1+     1:160      0.0694           2+    1:80        0.0578           2+    1:40        0.1198           3+    1:20        0.2236           4+    1:10        0.5235           5+    1:5         0.8833    ______________________________________

An UV inactivated antigen preparation from the Long Strain of RSV waspurchased and contained a total of 2×10⁵ PFU. This antigen preparationwas diluted in buffer, 1:1, and assayed as described above. 15 μl ofeach sample was applied to the surface and represented 7.5% of the totalantigen available.

                  TABLE 24    ______________________________________    RSV Standard Curve    Antigen Conc.                 Ellipsometric Intensity (mV)    ______________________________________    0            0.0079    1 × 10.sup.5                 1.0221    1 × 10.sup.4                 0.3124    2 × 10.sup.3                 0.0466    ______________________________________

EXAMPLE 26

Wavelength Dependence For A Thin Film Analyzer

Lundstrom, et al., U.S. Pat. No. 4,521,522, describe an instrument inwhich an incoming light beam passes through a polarizer and is reflectedoff the film at Brewster's angle for the substance to be measured. Theincident radiation is polarized parallel to the plane and angle ofincidence. The instrument requires the use of only one polarizer,however, when a second polarizer is used it must be set to exactly 900relative to the first polarizer. A single polarizer is all that isrequired as there is no change in the polarization of the incident lightupon reflection. For this polarization there is no change in thepolarization for any incidence angle, demonstrated by the fact that thepolarizer may be situated in either the incident or reflected beams.Only that part of both the light incident on the sample and thereflected light which is polarized parallel to the plane of incidence ismeasured.

The method is based on the minimum in reflectance observed at this anglewhen light polarized parallel to the plane of incidence is incident onthe interface between dielectric media. This phenomena is only observedat Brewster's angle. A substrate with a high refractive index isrequired. A metal, which is highly reflective, but has a low refractiveindex has too shallow a reflection minimum for this technique.

For better signal resolution of small thickness changes, the abovemethod requires a layer of oxide over the substrate to shift the minimumreading to an acceptable position in the reflection curve observed forpolarized light, allowing a small thickness change to produce a largerchange in the reflected light intensity. When an attachment layer isincluded, it is included for optimization of the substrate relative tothe reflectance minimum, and not to increase performance of thereceptive material.

With the pure parallel polarized light (p-polarized) described byLundstrom a very minor change in the % reflectivity is observed withanalyte binding; there is no change in the polarization of the light. Inthe current invention, in addition to the p-polarized light, there arealso components of perpendicularly polarized light (s-polarized) in theincident light beam. Very large changes in rotation of the polarizedlight are observed upon interaction with thin films and these changes inthe polarization rotation are measured as well as any change inintensity.

For very large angles of incident light, using the current invention,some degree of ellipticity can be generated in the light reflected fromthe test surface. The current invention maximizes a change inpolarization rotation upon reflection with added mass, and is notassociated with a minimum in reflectivity. The rotation of polarizationupon reflection observed with the current invention is entirely absentfor pure parallel polarized light. An additional advantage of thecurrent invention is that nearly any substrate and dielectric thin filmmay be used, since it does not rely on the Brewster angle. Also theangle of incident light is not critical and does not have to be changedwith the type of substrate material.

The current invention uses a monochromatic, collimated, light sourcesuch as a laser. It also includes a polarizer between the light sourceand the test surface oriented such that both p- and s-polarizedcomponents are incident on the surface. If the light source issufficiently well polarized this polarizer is unnecessary. The lightreflected from the surface passes through a second polarizer (theanalyzer) and enters a photodiode. The fraction of s-polarized light isselected to maximize the signal change as a function of thickness whilemaintaining a low background light intensity. The light becomeselliptically polarized upon reflection, with the ellipticity and angleof rotation of polarization depending in a very sensitive manner on theoptical properties of the surface. In a comparison ellipsometer, such asdescribed in U.S. Pat. Nos. 4,332,476, 4,665,595, and 4,647,207, thelight is then reflected off a reference surface which cancels thatellipticity in regions where the reference surface is opticallyidentical to the sample surface. The current invention does not requirea reference surface because it is not designed to establish the exactphysical thickness or refractive index of a given material. Instead, theintensity of the light passed through the analyzer provides a measure ofthe polarization rotation concomitant with the ellipticity, and hence arelative measure of optical properties of the surface. Thus, it providesa simple means to measure the change in the thickness and refractiveindex of the surface materials.

The measurement protocol is essentially unmodified from the instrumentabove using x, y positioning platforms to determine where readings aremade. However, the analyzing polarizer must be rotated to a pre-selectedvalue for the background prior to making an initial reading. Thepreferred embodiment includes a polarizer near the analyzer. Light istransmitted through the analyzer to a detector. Readings may be recordedin volts or millivolts. Readings may be displayed on an LED or otherdisplay device or captured by a data processing package.

The impact of thin films on the lightness and wavelength dependence oflight reflected from silicon was modeled with a computer simulation. Theresponse of a thin film with a refractive index of 1.459 was modeledusing a silicon surface and varying the thickness from 0 to 12 nm. Thewavelength ranges examined were 400-420 nm, 540-560 nm, and 680-700 nm.An increase in lightness from the minimum lightness to the maximumlightness represents an increase in light intensity as a function of anincrease in thickness. The lightness is a logarithm function of lightintensity. From this data, the maximum sensitivity change as a functionof thickness was achieved with a 540-560 nm light source. These datawere generated using a 70° angle of incidence and the values will changeslightly with the angle of incidence.

                  TABLE 25    ______________________________________    Wavelength              Min. Lightness                            Max. Lightness                                       Change    ______________________________________    400-420   -6.5          -4.5       -2.0    540-560   -4.2          -1.7       -2.5    680-700   -6.0          -4.3       -1.7    ______________________________________

To confirm these observations, a standard antigen dilution curve wasgenerated using the 9 minute assay protocol described in example 28.Intensities from the antigen dilution curve were measured, using thephotodiode modified Comparison Ellipsometer, as a function of incidentlight wavelength. As the Thin Film Analyzer is a simplification of theComparison Ellipsometer similar results are anticipated. Varyingwavelengths of incident light were achieved by filtering white lightthrough a narrow bandpass filter to select specific wavelengths. Allfilters were Corin P70 series filters.

                  TABLE 26    ______________________________________    Incident Light                  Antigen Dilution                              mV Response    ______________________________________    White         0           310                  1:9600      317                  1:4800      319                  1:2400      371                  1:1600      483                  1:1000      780                  1:400       1128    550 nm        0           28                  1:9600      29                  1:4800      30                  1:2400      36                  1:1600      56                  1:1000      93                  1:400       176    600 nm        0           33                  1:9600      33                  1:4800      34                  1:2400      42                  1:1600      66                  1:1000      113                  1:400       221    450 nm        0            9                  1:9600       9                  1:4800       9                  1:2400      11                  1:1600      17                  1:1000      27                  1:400       50    ______________________________________

EXAMPLE 27

Thin Film Analyzer Angle Dependence

The thin film analyzer shown in FIG. 14a was used with a 672 nm diodelight source and the dual polarizer set-up diagrammed. The laser diodeand photodiode were both mounted to accurately assess the angle ofincidence and detection relative to a normal angle of incidence. Strep Aantigen dilution curves were used to examine the angle dependence of theanalyzer. The detector used with the thin film analyzer prematurelysaturates, actual dynamic range is 3500+mV. For a reference, the antigencurves were also examined with a small comparison ellipsometer alsoequipped with a 672 nm laser diode source.

                  TABLE 27    ______________________________________    Comparison Ellipsometer Readings    Antigen Dilution                Average mV   3SD    Range    ______________________________________    0           160.3        9.69   150.6-170.0    1:9600      190.4        5.58   184.8-195.9    1:2400      367.4        15.9   351.1-383.3    1:1000      766.0        26.3   739.7-792.3    1:400       1623.7       1.4    1622.3-1625.1    ______________________________________

                  TABLE 28    ______________________________________    Thin film Analyzer    Angle Antigen Dilution                       Average mV.sup.a                                  3SD   Range    ______________________________________    30*   0            12.0          1:9600       68.0          1:2400       90.0          1:1000       188.0          1:400        204.0    40*   0            64.0          1:9600       78.0          1:2400       84.0          1:1000       197.0          1:400        343.0    50*   0            221.3      7.95  213.4-229.3          1:9600       263.4      13.2  250.2-276.6          1:2400       370.9      28.2  342.7-399.1          1:1000       574.2      54.0  520.2-628.2          1:400        1063.7     76.8   986.9-1140.5    56*   0            133.3      9.0   124.3-142.3          1:9600       158.0      11.7  146.3-169.7          1:2400       340.1      44.4  295.7-384.5          1:1000       554.8      110.1 444.7-664.9          1:400        1025.2     84.6   940.7-1109.8    60*   0            308.9      18.6  290.3-327.5          1:9600       352.3      9.6   342.7-361.9          1:2400       569.9      36.6  533.3-606.5          1:1000       1070.7     159.0 911.7-1229.7          1:400        1651.0     0.0   1651.0    70*   0            1300.0          1:9600       1372.0          1:2400       1482.0          1:1000       1649.0          1:400        1649.0    ______________________________________     .sup.a Average mV signal from ten separate readings.     *Data represents single measurement at these angles.

At angles of incidence in the range of 300 to 400, the thin filmanalyzer demonstrates excellent sensitivity, and the dynamic rangeobserved is suitable for assays with a limited range requirement. Theangles between 50° and 60° meet both the requirements of sensitivity anddynamic range. Background cannot be sufficiently minimized with a singlepolarizer for angles above 65°. This background could be reduced byelectronic means. The dynamic range at these angles is sufficient toallow such correction mechanisms.

EXAMPLE 28

Comparison Ellipsometer Sensitivity For Strep A Assay

A Strep A assay protocol was developed which further enhances thereadability of low positive samples. The sample (Strep A antigen) wasmixed with conjugate and incubated 3 minutes at room temperature. Analiquot (20 μl) of this mixture was applied to the surface and incubatedfor 3 minutes. The rinse/dry protocol was performed as described above,either in the single use device or under a stream of nitrogen. Substratewas applied and incubated for 3 minutes, followed by the rinse/dryprotocol. Results for an antigen dilution curve prepared with testsurfaces with and without (visual--versus--non-visual) silicon nitride.Visual results were scored for increasing hues of purple from palepurple to dark blue. Surfaces without silicon nitride were reacted,rinsed, and dried without incorporation into a single use device. Thesesurfaces were examined with a photodiode modified ComparisonEllipsometer. Readings were reported in millivolts and corrected for abackground measurement. An eight fold increase in the performance of theStrep A OIA assay was observed with the instrumented detection based onan antigen dilution study. A total of 5 curves were examined.

                  TABLE 29    ______________________________________    Antigen Dilution               Visual   Average mV 3SD   Range    ______________________________________    0          -        2.5        3.0   -0.5-5.5     1:38400   -        16.8       6.6   10.2-23.4     1:19200   -        38.4       12.3  26.1-50.7    1:9600     -        54.5       11.7  42.8-66.2    1:4800     1+       161.3    1:2400     2+    1:1600     5+    1:1000     7+    1:400      10+    ______________________________________

The test kit, the immunoassay device and the underlying coating anddetection methods described herein are not intended to be limited by theassay format described or by the volumes, the concentrations or specificingredients given for the various reagents, controls, and calibrators.It should be understood that similar chemical or other functionalequivalents of the components used in the layer, layer coatings, or inany of the various reagents, additives, controls, and calibrators can beutilized within the scope of this invention.

The foregoing examples serve to illustrate the efficiency and utility ofthis technology to detect a variety of analytes using the pre-formedslide consisting of a substrate, AR material(s), activation, andreceptive material(s) to produce an interference color change as asignal of analyte attachment.

Without being bound to the substrate formats or materials utilized inthe preceding examples, it is possible to utilize a diversity ofcombinations of substrate formats and substrate materials which arefunctionally equivalent substitutes capable of having AR material boundto their surface, or are capable of being activated to allow attachmentof the receptive material.

It is contemplated that the inventive concepts herein described may havediffering embodiments and it is intended that the appended claims beconstrued to include all such alternative embodiments of the inventionexcept insofar as they are limited by the prior art.

We claim:
 1. Method for the determination of chlamydial or gram negativebacterial antigen comprising:providing an optically active surfacecomprising an attachment layer and a layer of non-specific protein, saidoptically active surface exhibiting a first color in response to lightimpinging thereon, and exhibiting a second color comprising an intensityof at least one wavelength of light different from said first color, inresponse to said light when said antigen is present on said attachmentlayer, said attachment layer selected from the group of chemicalsconsisting of dendrimers, star polymers, molecular self-assemblingpolymers, polymeric siloxanes, and film forming latexes, provided onsaid optically active surface, which promote adhesion of the antigen tothe optically active surface by hydrophobic interactions, and said layerof non-specific protein provided on said attachment layer, contactingsaid attachment layer with a sample potentially containing extractedantigen under conditions in which said antigen can bind to saidattachment layer and can interact with said optically active surface tocause said optically active surface to exhibit said second color whensaid antigen is present, and detecting said second color by use of anellipsometer, a reflectometer, a comparison ellipsometer, a profilometeror a thin film analyzer as an indication of the presence of saidantigen.
 2. The method of claim 1, wherein the antigen is an LPS(Lipopolysaccharides).
 3. The method of claim 1, wherein the antigen isa major outer membrane protein.
 4. Method for determination of achlamydial or gram negative bacterial antigen consisting of:mixing asample suspected of containing an analyte of interest with a reagent toextract antigens and allowing this extraction to proceed for 5 to 10minutes, and adding sufficient volume of a neutralizing buffer to adjustthe final pH to 7.0-7.5, and applying the sample to an optically activesurface comprising an attachment layer and a layer of non-specificprotein for 5 to 10 minutes at room temperature, said optically activelayer exhibiting a first color in response to light impinging thereon,and exhibiting a second color comprising an intensity of at least onewavelength of light different from said first color, in response to saidlight when said antigen is present on said attachment layer, saidattachment layer selected from the group of chemicals consisting ofdendrimers, star polymers, molecular self-assembling polymers, polymericsiloxanes, and film forming latexes, provided on said optically activelayer, which promote adhesion of the antigen to the optically activesurface by hydrophobic interactions, said layer of non-specific proteinprovided on said attachment layer, applying a antibody conjugated to anenzyme to the sample on the optically active surface for 1 to 10minutes, followed by a wash and dry protocol, and applying substrate tothe optically active surface for 10 to 15 minutes at room temperature,which can react with said enzyme portion of said antibody conjugatebound to said antigen bound to said attachment layer to form a productdeposited on said bound antigen, followed by a wash and dry protocol,and detecting the second color produced by said antigen and saidproduct.
 5. The method of claim 4, wherein the color change is detectedwith an ellipsometer.
 6. The method of claim 4, wherein the color changeis detected with a comparison ellipsometer.
 7. The method of claim 4,wherein the color change is detected with a thin film analyzer.
 8. Themethod of claim 4, wherein the color change is detected with areflectometer.
 9. The method of claim 4, wherein the extraction reagentconsists of phosphate buffered saline containing approximately 0.1%chenodeoxycholic acid (CDOC) and is alkalized with NaOH to a final pH ofapproximately 11.5.
 10. The method of claim 4, wherein the neutralizingbuffer is a phosphate buffer.
 11. Method for the determination ofchlamydial or gram negative bacterial antigen comprising:providing anoptically active surface comprising an anti-reflective film, anattachment layer and a layer of non-specific protein, said opticallyactive surface exhibiting a first color in response to light impingingthereon, and exhibiting a second color comprising a combination ofwavelengths of light different from said first color, in response tosaid light when said antigen is present on said attachment layer, saidanti-reflective film provided on said optically active surface, saidattachment layer selected from the group of chemicals consisting ofdendrimers, star polymers, molecular self-assembling polymers, polymericsiloxanes, and film forming latexes, provided on said anti-reflectivefilm, which promote adhesion of the antigen to the optically activesurface by hydrophobic interactions, said layer of non-specific proteinprovided on said attachment layer, contacting said attachment layer witha sample potentially containing extracted antigen under conditions inwhich said antigen can interact with said optically active surface tocause said optically active surface to exhibit said second color whensaid antigen is present, and visually detecting said second color. 12.The method of claim 11, wherein said antigen is an LPS(Lipopolysaccharide).
 13. The method of claim 11, wherein the antigen isa major outer membrane protein.
 14. Method for the determination ofchlamydial or gram negative bacterial antigen comprising:providing anattachment layer selected from the group of chemicals consisting ofdendrimers, star polymers, molecular self-assembling polymers, polymericsiloxanes, and film forming latexes, which promote adhesion of theantigen to said attachment layer by hydrophobic interactions, and alayer of non-specific protein provided on said attachment layer,contacting said attachment layer with a sample potentially containingextracted antigen under conditions in which said antigen can bind tosaid attachment layer, contacting said antigen bound to said attachmentlayer with a reagent able to specifically bind to said bound antigen andable to generate a signal, and detecting said signal as an indication ofthe presence of said antigen.
 15. The method of claim 14, wherein saidsignal generated is from an ELISA.
 16. The method of claim 14, whereinsaid signal generated is from an RIA.
 17. The method of claim 14,wherein the signal generated is from a fluorescent or chemiluminescentlabel.
 18. Method for determination of a chlamydial or gram negativebacterial antigen consisting of:mixing a sample suspected of containingan analyte of interest with a reagent to extract antigens and allowingthis extraction to proceed for 5 to 10 minutes, and adding sufficientvolume of a neutralizing buffer to adjust the final pH to 7.0-7.5, andapplying the sample to an optically active surface comprising, ananti-reflective film, an attachment layer and a layer of non-specificprotein for 5 to 10 minutes at room temperature, said optically activelayer exhibiting a first color in response to light impinging thereon,and exhibiting a second color comprising a combination of wavelengths oflight different from said first color, in response to said light whensaid antigen is present on said attachment layer, said anti-reflectivefilm provided on said optically active layer, said attachment layerselected from the group of chemicals consisting of dendrimers, starpolymers, molecular self-assembling polymers, polymeric siloxanes, andfilm forming latexes, provided on said anti-reflective layer, whichpromote adhesion of the antigen to the optically active surface byhydrophobic interaction said layer of non-specific protein provided onsaid attachment layer, applying an antibody conjugated to an enzyme tothe sample on the optically active surface for 1 to 10 minutes, followedby a wash and dry protocol, and applying substrate to the opticallyactive surface for 10 to 15 minutes at room temperature, which can reactwith said enzyme portion of said antibody conjugate bound to saidantigen bound to said attachment layer to form a product deposited onsaid bound antigen, followed by a wash and dry protocol, and visuallydetecting the second color produced by the presence of said antigen andsaid product on said attachment layer.